JP5711666B2 - Efficient transport to leukocytes - Google Patents

Description

  The present invention relates to the use of specific transporter-loading conjugate molecules to transport a substance of interest (a cargo molecule) to leukocytes. The transporter-loading conjugate molecule can be used to treat, prevent, attenuate, and / or ameliorate diseases and / or disorders involving leukocytes. The present invention also relates to the production of the transporter-loading conjugate molecule, a method for transporting a target substance (loading) to leukocytes, and a leukocyte comprising the transporter-loading conjugate molecule or a fragment thereof.

  Technologies that can efficiently transport not only target substances such as nucleic acids, proteins, or cytotoxic agents, but also other compounds from external media to tissues or cells, especially cell nuclei, are of great interest in the field of biotechnology. . These techniques transport and translate nucleic acids into cells in vitro and in vivo, thereby producing proteins or (poly) peptides, controlling gene expression, inducing cytotoxic or apoptotic effects, analyzing intracellular processes, And may be suitable for analysis of effects produced by transporting various different loads to cells (or cell nuclei).

  One important application for transporting a subject load from an external medium to a tissue or cell is targeted drug delivery. Said targeted drug delivery relates to tissue / cell specific delivery of a pharmaceutically active agent. The intention is to increase the concentration of the pharmaceutically active agent in the target cells and tissues over time after (systemic) administration, while maintaining a low level of the pharmaceutically active agent in the remaining cells and tissues. It is done. The targeted drug delivery can enhance therapeutic effects and reduce side effects. The targeted drug delivery can be mediated, for example, by highly specific antibodies.

  A further important use of transporting a target load from an external medium to a tissue or cell is gene therapy, where the load is generally a nucleic acid or a gene. This technology has undergone some very promising developments over the past decades. However, gene transfer generally involves biology of the gene transfer vector into the cytoplasm or nucleus of the host cell being treated without affecting the host genome or changing the biological properties of the active cargo. Is subject to the limitation that efficient active loads cannot be transported efficiently.

  In this regard, several techniques have been developed to more efficiently transfect cells with, for example, nucleic acids such as DNA or RNA. Transfection of nucleic acids into patient cells or tissues by gene delivery is a core method of molecular medicine and plays an important role in the treatment and prevention of many diseases.

  Examples of gene transport methods include general (physical or physicochemical methods) such as a method in which nucleic acid is coprecipitated with calcium phosphate or DEAE-dextran, and a method in which nucleic acid is allowed to pass through a cell membrane and then enter a cell and / or nucleus. ) Method. However, this technique has the problems of low transport efficiency and a high cell death rate. Furthermore, this method is not inherently applicable to in vivo methods and is limited to in vitro methods or ex vivo methods.

  The same is true for in vitro electroporation methods. The in vitro electroporation method is a method of introducing a new nucleic acid such as DNA or RNA into a cell by imparting a high voltage current to the cell membrane to make the cell membrane permeable. However, such methods are generally not suitable for in vivo. Furthermore, this technique also has the problems of low transport efficiency and high cell death rate.

  Furthermore, well-known physical or physicochemical methods include (direct) injection of (naked) nucleic acids or gene transport with a gene gun. Gene transport by gene gun (also known as gene gun particle bombardment) is a method of introducing genetic material into tissues or cultured cells developed at Cornell University. In general, gene transport using a gene gun is performed by applying a surface coating to metal particles such as gold or silver particles, and shooting these metal particles containing adsorbed DNA into cells using a gene gun. As above, this method is also limited to in vitro or ex vivo methods, but is generally not applicable in in vivo situations.

  Another method utilizes the transport ability of so-called transporter molecules. Transporter molecules used in this context can generally be classified into viral vectors containing viral elements, ie transporter molecules and non-viral vectors.

  The most successful of the gene therapy strategies available today is gene therapy that relies on viral vectors such as adenovirus, adeno-associated virus, retrovirus, and herpes virus. These viral vectors generally use conjugates of virus-related substances that have a strong affinity for DNA and nucleic acids. Due to its infectivity, the virus or viral vector has a very high transfection efficiency. Commonly used viral vectors are genetically modified so that functional infectious particles are not formed in the transfected cells. However, despite the safeguards from these genetic modifications, there are many problems associated with viral vectors regarding immunogenicity, cytotoxicity, and insertional mutagenesis. An example of this is the problem that the risk of recombination events occurring and uncontrolled growth of the introduced therapeutically active gene or viral gene cannot be avoided. Furthermore, virus conjugates are difficult to use and generally require long-term preparation before treatment (see, for example, Patent Document 1).

  Non-viral vectors are not as efficient as viral vectors when used for gene therapy, but many have been developed to provide safer alternatives in gene therapy. Some of the most common non-viral vectors include transport systems based on polyethyleneimine, dendrimers, chitosan, polylysine, and (poly) peptides, such as many types of (poly) peptides, which are generally essentially It is cationic in nature and can interact with nucleic acids such as plasmid DNA through electrostatic interactions. In addition, non-viral vectors can perform drug delivery that is not based on nucleic acids.

  In order for the drug delivery to be successful, non-viral vectors, particularly transport systems based on (poly) peptides, must be able to overcome many problems. Such problems include, for example, protecting cargo molecules such as DNA or other compounds during transport and preventing initial degradation or metabolism of the cargo molecule in vivo. In the case of nucleic acids such as DNA and RNA molecules, non-viral vectors also need to specifically deliver the molecules in order to efficiently express genes in target cells.

  In particular, with respect to nucleic acids such as DNA and RNA molecules, there are currently four problems that non-viral vectors must overcome in order to achieve successful gene delivery (see, for example, Non-Patent Document 1). That is, non-viral vectors: 1) densely compress and protect nucleic acids, 2) target specific cell surface receptors, 3) disrupt endosomal membranes, and 4) use nucleic acid cargo as a nucleus There are four problems of being able to deliver and to translate the encoded protein or (poly) peptide sequence.

  Such non-viral vectors, particularly non-viral vectors based on (poly) peptides, are generally beneficial over other non-viral vector strategies in that they can achieve all four objectives, but differ in terms of efficiency. There is a problem.

  For example, cationic (poly) peptides rich in basic residues such as lysine and / or arginine efficiently condense nucleic acids such as DNA into small dense particles that can be stabilized in serum. Can do. Furthermore, specific receptors and / or specific cell types can be targeted by binding (poly) peptide ligands to the polyplex. Polyplexes or cationic polymers as described above generally form complexes with negatively charged nucleic acids to condense the nucleic acids and protect these nucleic acids from degradation. Transport to cells using polyplexes (cationic polymers) generally occurs via receptor-mediated endocytosis. Thereby, the DNA binds to different molecules such as transferrin, for example via polyplex poly-L-lysine (PLL) which binds to surface receptors and induces endocytosis. Examples of the polyplex (cationic polymer) include poly-L-lysine (PLL), chitosan, polyethyleneimine (PEI), polydimethylaminoethyl methacrylate (PD-MAEMA), and polyamidoamine (PAMAM). Such an effect is also known for nanoplex (nanoparticle system) or lipoplex (liposome system). Nanoplexes (nanoparticle systems) generally include the use of polyacrylates, polyamides, polystyrenes, cyanoacrylates, polylactic acid (PLA), lactic acid-glycolic acid copolymers (PLGA), and the like. Lipoplexes, ie liposome systems, generally involve the use of cationic lipids that can mimic cell membranes. Thereby, the positively charged portion of the lipid can interact with the negatively charged portion of the nucleic acid and fuse with the cell membrane. Examples of the lipoplex, that is, the liposome system, include DOTMA, DOPE, DOSPA, DOTAP, DC-Chol, EDMPC, and the like.

  In this situation, receptor-mediated endocytosis has also been extensively studied in experimental systems for targeted delivery of cargo or therapeutic agents such as nucleic acids to cells. During receptor-mediated endocytosis, cargo-containing complexes are selectively internalized by specific receptors of cargo located at the cell membrane or by specific antibodies located within membrane components. Endocytotic activity has been reported for many receptors including IgG Fc, somatostatin, insulin, IGF-I, IGF-II, transferrin, EGF, GLP-1, VLDL, or integrin receptors.

  Various (poly) peptide or protein sequences have been extensively tested for use in gene delivery methods via the receptor-mediated endocytosis. Interestingly, the isolation of (poly) peptide sequences leading to efficient receptor-mediated endocytosis has made significant progress by using phage display technology. The phage display library is a very powerful tool that provides an almost infinite number of molecular mutagens, including natural ligands or cargo molecules for cell receptors and short (poly) peptide modifications. In addition, a similar library has been successfully injected directly into mice to successfully isolate (poly) peptide sequences that exhibit 13-fold selectivity for brain and kidney.

  Proprotein converting enzymes can be mentioned as examples of (poly) peptide or protein sequences that can be used to transport molecules into cells. The proprotein converting enzyme is an example of a cell surface receptor that is internalized via receptor-mediated endocytosis. These proteins have been shown to be involved in the conversion of (poly) peptide hormones, neuropeptides, and many other protein precursors to biologically active forms. All cleavage sites of the proprotein converting enzyme family have the consensus sequence R—X—X—R. Mammalian proprotein converting enzymes can be divided into three groups based on tissue distribution. Furin, PACE4, PC5 / PC6, and LPCIPC7 / PC8 / SPC7 are expressed in a wide range of tissues and cell lines. In contrast, PC2 and PC1 / PC3 expression is restricted to neuroendocrine tissues such as the islets of Langerhans, the pituitary gland, the adrenal medulla, and many brain regions. PC4 expression is very limited to testicular sperm cells. PC2 and PC1 / PC3, which are neuroendocrine-specific converting enzymes, are mainly localized in secretory granules. PC5 / PC6A has also been reported to localize in secretory granules. Furthermore, indirect evidence that some of the proprotein converting enzyme molecules are present on the cell surface has been shown, indicating that furin circulates between TGN and the cell surface. Taken together, these properties indicate that proprotein converting enzymes transport extracellular ligands into the intracellular space.

  Also advantageous are so-called translocation proteins or protein transduction domains (PTD). (Poly) peptide sequences derived from transport proteins or protein transduction domains (PTDs) are generally capable of selectively lysing endosomal membranes in an acidic environment that leads to cytoplasmic release of the polyplex. Transport proteins such as HIV-1 TAT (HIV), Antennapedia (Drosophila antennapedia), HSV VP22 (herpes simplex), FGF, or lactoferrin can perform macromolecular transport between cells (poly) peptide ( A group of transport proteins). In contrast, protein transduction domains (PTDs) are thought to be a group of (poly) peptides that can direct proteins and (poly) peptides covalently bound to these sequences to cells through the cell membrane ( Non-patent document 2). Transport proteins and PTDs share a common basic region, which is thought to be primarily involved in the transport of fusion (poly) peptides because it can bind to polyanions such as nucleic acids. . Without being bound by this theory, PTD can show similar effects to cationic transfection reagents that use receptor-dependent unsaturated adsorption endocytosis. PTDs generally bind to proteins or (poly) peptides to affect or enhance CTL responses when administered (poly) peptide-based vaccines (reviews, (Refer nonpatent literature 3).

  In general, several techniques are known in the art that can transport a substance of interest from an external medium to a tissue or cell, but there is a need for techniques that can be efficiently transported to specific tissues and cell types. Sex is still high.

US Pat. No. 5,521,291

Martin et al. , The AAPS Journal 2007; 9 (1) Article 3 Leifert and Whitton: Transplantation proteins and protein transduction domains: Molecular Therapy Vol. 8 No. 1 2003 Melikov and Chernomordik, Arginine-rich cell penetrating (poly-) peptides: from endosomal uptake to nuclear delivery, Cell. Mol. Life Sci. 2005

  Accordingly, it is an object of the present invention to provide a novel means of targeting specific tissues and cell types efficiently and with high specificity, for example, loads such as drugs and effector molecules.

  This problem is solved for white blood cells (WBC) by using the inventive subject matter described in the appended claims. Specifically, the inventors of the present application have surprisingly found that certain types of (poly) peptides efficiently transport the cargo of interest to leukocytes.

FIG. 1 shows in vitro FITC-labeled TAT-derived transporter constructs (0 nmol / L (nM), 100 nmol / L (nM), or 500 nmol / L (nM)) of HepG2 hepatocellular carcinoma cells, HCT-116 neoplastic colon Cells and uptake (internalization) into U937 lymphoma cells are shown. The construct used was FITC-labeled D-TAT (SEQ ID NO: 4). FITC-labeled D-TAT was internalized more in the WBC strain (U937; lymphoma) than in the non-WBC strain (HepG2: hepatocellular carcinoma cell; HCT-116: neoplastic colon cell). FIG. 2 shows the uptake (internalization) of FITC-labeled TAT-derived transporter construct (1 μmol / L (μM)) over time in vitro. Three different cell lines were used (HepG2 hepatocellular carcinoma cells, A549 lung carcinoma cells, and J77 macrophage cells). The construct used was FITC-labeled D-TAT (SEQ ID NO: 4). FITC-labeled D-TAT was internalized more in the WBC strain (J77 macrophage cells) than in the non-WBC strain (HepG2; A549). FIG. 3 shows the results of time-dependent internalization (incorporation) of FITC-labeled TAT-derived transporter construct into cells of the HL-60 cell line. The transporter-loading conjugate molecules used were FITC-labeled DAK (SEQ ID NO: 232), FITC-labeled L-TAT (SEQ ID NO: 2), FITC-labeled r3-L-TAT (SEQ ID NO: 15), FITC-labeled r3-L- TATi (SEQ ID NO: 16) and FITC-labeled D-TAT (SEQ ID NO: 4). HL-60 cells were incubated with 10 mol / L (M) TAT derivative transporter for 30 minutes, 1 hour, 6 hours, or 24 hours. The cells were then washed twice with acidic buffer (0.2 mol / L (M) glycine, 0.15 mol / L (M) NaCl, pH 3.0) and twice with PBS. Cells were disrupted by the addition of RIPA lysis buffer. Then, after reading the fluorescence intensity of each extract (Fusion Alpha plate reader; PerkinElmer), the relative amount of internalized peptide was determined by reducing the background and normalizing the protein content. The r3-L-TAT transporter construct showed internalization ability as effective as the D-TAT transporter construct. FIG. 4 shows uptake of FITC-labeled TAT-derived transporter construct (internalization) in vitro (10 μmol / L (μM), U937, lymphoma, 24 hours). The constructs used were four different TAT-derived transporter constructs (L-TAT (SEQ ID NO: 2), r3-TAT (also called r3-L-Tat) (SEQ ID NO: 15), r3-TATi (r3-L-TATi). (Also referred to as) (SEQ ID NO: 16), and D-TAT (SEQ ID NO: 4)), each having an amino acid length of 9, but having a different D- / L-pattern. In addition, a construct DAK (SEQ ID NO: 232) containing only amino acids D, A, and K was used as a control sample for comparison. As can be seen, uptake of transporter constructs into cells in r3-TAT (SEQ ID NO: 15), r3-TATi (SEQ ID NO: 16), and D-TAT (SEQ ID NO: 4) is the most efficient, -There was little uptake of TAT (SEQ ID NO: 2) into cells. FIG. 5 shows the co-localization between FITC-D-TAT labeled cells and CD-14 positive cells. (A): Primary cultured macrophages were incubated with XG-102 (SEQ ID NO: 233) and washed vigorously. The presence of XG-102 (SEQ ID NO: 233) was revealed using a specific antibody against XG-102. XG-102 was highly incorporated into primary macrophages. (B): 0.1 mg / kg of FITC-D-TAT (SEQ ID NO: 4 + FITC) was injected intravenously into the mice, and 24 hours later, the mice were sacrificed by perfusing the heart with PAF. Sections were prepared after cryoprotection using a cryostat. Immunostaining for the CD-14 receptor (present on the cell surface of macrophages and granulocytes) showed co-localization between FITC-D-TAT labeled cells and CD-14 positive cells (liver resident macrophages). Are called Kupfer cells). FIG. 6 shows the uptake (internalization) of FITC-labeled TAT-derived transporter construct (SEQ ID NO: 4) in 11 different WBC strains (10 μmol / L (μM)). All WBC strains tested internalized D-TAT-FITC in a time-dependent manner. FIG. 7 shows the effect of TAT on tissue distribution. Three different routes of administration (subcutaneous, intravenous, intraperitoneal) mice were treated with radiolabeled peptides (1 mg / kg) at C ¹⁴ through. Animals were sacrificed 72 hours after injection and processed for immunoradiography. The sagittal section was exposed and revealed that three different D-TAT tagged peptides accumulated mainly in the liver, spleen, and bone marrow (D-TAT: SEQ ID NO: 4; XG-102: SEQ ID NO: 233). XG-414: D-TAT bound to Bok's dBH3 domain). This indicates that the transporter D-TAT is involved in tissue distribution. FIG. 8 shows that FITC-D-TAT (SEQ ID NO: 4) is significantly taken up by Kupffer cells. Mice were injected intravenously with 0.1 mg / kg FITC-D-TAT (SEQ ID NO: 4) and sacrificed by perfusion 24 hours later. The liver was excised, cryopreserved and cut into sections in a cryostat. D-TAT was made directly visible via FITC fluorescence and cell nuclei were labeled with Hoechst dye. Liver parenchymal cells are giant cells that exhibit large nuclei (89% of liver cells), while Kupffer cells (liver resident macrophages) make up about 10% of the cells and are small, thin, elongated with small nuclei. It is a cell. Based on morphology, it can be concluded that FITC-D-TAT (SEQ ID NO: 4) accumulates primarily in Kupffer cells. FIG. 9 shows immunostaining of XG-102 (SEQ ID NO: 233) in the liver of rats injected intravenously with 1 mg / kg XG-102. Animals were sacrificed 24 hours after injection. Color development was performed using DAB substrate. This figure again shows a marked accumulation of XG-102 in the liver, especially in Kupffer cells. FIG. 10 is treated with XG-102 (SEQ ID NO: 233) in an IBD experiment (IBD: inflammatory bowel disease) that is subcutaneously treated once daily with XG-102 at concentrations of 1 μg / kg and 100 μg / kg. The clinical score is shown. FIG. 11 is subcutaneously treated once daily with XG-102 at concentrations of 0.01 μg / kg, 0.1 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, and 1,000 μg / kg. In the IBD experiment, a dose response curve when treated with XG-102 (SEQ ID NO: 233) is shown. FIG. 12 shows XG-102 (SEQ ID NO: 233) in an IBD experiment treated with XG-102 at a concentration of 1 μg / kg and 100 μg / kg as a single dose on day 0 (single dose SC). Shows clinical score when processed. FIG. 13 shows that when treated with XG-102 (SEQ ID NO: 233) in an IBD experiment treated orally with XG-102 at concentrations of 1 μg / kg and 100 μg / kg as repeat doses (once daily PO). The clinical score is shown. FIG. 14 shows XG-102 (SEQ ID NO: 233) in an IBD experiment treated on day 0 with XG-102 at a concentration of 1 μg / kg and 100 μg / kg as a single dose (single dose PO). Shows clinical score when processed. FIG. 15 shows that the fluorescent TAT derivative transporter D-TAT (SEQ ID NO: 4) -FITC or r3-L-TAT (SEQ ID NO: 15) -FITC targets different human leukocyte populations. FIG. 15A: D-TAT (SEQ ID NO: 4) -FITC. The percentage of cells entering and exiting each quadrant is as follows (clockwise from the upper left quadrant): monocytes (CD14) 9.24; 19.37; 31.71; 39.68; CD15) 17.03; 13.87; 21.53; 47.57; lymphocyte T (CD3) 22.7; 11.82; 18.01; 47.46; lymphocyte B (CD19) 32.40; 2.12; 8.26; 57.22. FIG. 15B: r3-L-TAT (SEQ ID NO: 15) -FITC. The percentage of cells entering and exiting each quadrant is as follows (clockwise from the upper left quadrant): monocytes (CD14) 6.34; 16.65; 36.24; 40.77; CD15) 11.74; 13.75; 24.76; 49.75; lymphocyte T (CD3) 20.64; 8.96; 20.04; 50.36; lymphocyte B (CD19) 27.83; 1.76; 8.48; 61.92. FIG. 16 shows the average fluorescence value of fluorescent TAT derivative transporter D-TAT (SEQ ID NO: 4) -FITC or r3-L-TAT (SEQ ID NO: 15) -FITC in each cell type shown in FIG. 15 (FITC channel). Show. FIG. 17 shows the uptake by various cell types of selected transporter constructs according to the present invention. Incorporation is normalized to D-TAT (SEQ ID NO: 4). Raw: macrophage cells (mouse; leukocyte cell line); J77: macrophage cells (mouse; leukocyte cell line); BMDM: bone marrow derived macrophages (mouse; purified primary leukocytes). ^* N = 2 independent experiments (duplex) (except for peptide number 64 with n = 1,2), ^** experiments with n = 2 (double) (n = 2, peptide number with 2) 64)), ^*** n = 1 experiment (dual). FIG. 18 shows the uptake by various cell types of selected transporter constructs according to the present invention. _Uptake, normalized to r 3 -L-TAT (SEQ ID NO: 15). Raw: macrophage cells (mouse; leukocyte cell line); J77: macrophage cells (mouse; leukocyte cell line); BMDM: bone marrow derived macrophages (mouse; purified primary leukocytes). ^* N = 2 independent experiments (duplex) (except for peptide number 64 with n = 1,2), ^** experiments with n = 2 (double) (n = 2, peptide number with 2) 64)), ^*** n = 1 experiment (dual). FIG. 19 shows immunohistochemical staining of limbs undergoing CFA-induced inflammation (4 hours). The upper panel represents hindlimb hematoxylin and eosin staining (H & E). From left to right, saline treated animals not treated with CFA (complete Freund's adjuvant), saline treated animals treated with CFA, and XG-102 (SEQ ID NO: 233) treated with CFA injection Is an animal. After CFA treatment, the muscle and epithelial layers appear to be less organized due to edema. The presence of XG-102 (SEQ ID NO: 233) positive cells was detected only in animals treated with XG-102 (upper right panel). As evidenced by the appearance of brown-stained cells only in CFA-XG-102 treated animals (lower right panel), XG-102 It became clear that there exists. 19A: 20 × magnification, FIG. 19B: 40 × magnification, FIG. 19C: 100 × magnification. FIG. 20 shows that XG-102 (SEQ ID NO: 233) co-localizes with certain leukocyte populations in inflamed CFA (complete Freund's adjuvant) treated animals. FIG. 20A represents hind limb sections of CFA-treated animals stained with three different dyes. The four upper panels represent the same location of the distal region at high magnification. Distribution of leukocyte markers (mainly granulocytes, macrophages, and monocytes) cd11b (first from the left), XG-102 (SEQ ID NO: 233, second from the left), and DAPI nuclear staining (third from the left) Show. The upper right panel represents a merged image of cd11b and XG-102 positive staining. The lower panel (higher magnification) represents another area of the hind limb with edema and focuses on some selected inflammatory cells (arrows) that clearly show leukocyte infiltration (cd11b positive cells). (See bottom left panel). The lower right panel represents a merged image of cd11b and XG-102 positive staining. FIG. 20B is an enlarged view of the lower right panel of FIG. 20A. FIG. 21 shows experiment on lymph nodes—XG-102 (SEQ ID NO: 233) detection using peroxidase development. FIG. 5 shows a composite image (upper figure: 10 × objective lens) of several lymph node regions showing different structures and cell types. From left to right, the export lymphatic hilum, myelin cord, and cortex. The two lower panels are an illustration of the myelin (left) and cortex (right) (40x objective, bar 20 μm). Background was detected in the presence of anti-XG-102 (SEQ ID NO: 233) antibody using sections of 24 hour saline infused animals. By immunostaining lymph nodes derived from rats that had been pre-injected with XG-102 (SEQ ID NO: 233), leukocytes (larger as shown by XG-102-containing vesicles, from 30 minutes to 28 days after injection). It was revealed that XG-102 (SEQ ID NO: 233) was present in some of the resident macrophages and circulating macrophages. FIG. 21A is a view showing a composite image of a lymph node region of a 24-hour saline-injected animal. Upper panel: objective lens 10 ×; bar 50 μm; lower panel: objective lens 40 ×; bar 20 μm. FIG. 21B is a diagram showing a composite image of a lymph node region of an XG-102-injected animal after 30 minutes. Upper panel: objective lens 10 ×; bar 50 μm; lower panel: objective lens 40 ×; bar 20 μm. FIG. 21C is a diagram showing a composite image of a lymph node region of an XG-102-injected animal after 24:00. Upper panel: objective lens 10 ×; bar 50 μm; lower panel: objective lens 40 ×; bar 20 μm. FIG. 21D is a diagram showing a composite image of a lymph node region of an XG-102-injected animal after 3 days. Upper panel: objective lens 10 ×; bar 50 μm; lower panel: objective lens 40 ×; bar 20 μm. FIG. 21E is a diagram showing a composite image of a lymph node region of an XG-102-injected animal after 7 days. Upper panel: objective lens 10 ×; bar 50 μm; lower panel: objective lens 40 ×; bar 20 μm. FIG. 21F is a diagram showing a composite image of a lymph node region of an XG-102-injected animal after 14 days. Upper panel: objective lens 10 ×; bar 50 μm; lower panel: objective lens 40 ×; bar 20 μm. FIG. 21G is a view showing a composite image of a lymph node region of an XG-102-injected animal after 27 days. Upper panel: objective lens 10 ×; bar 50 μm; lower panel: objective lens 40 ×; bar 20 μm. FIG. 22A shows the localization of FITC-XG-102 (SEQ ID NO: 233) in the liver. It is a figure which shows three cell visual fields of the FITC-XG-102 labeled cell isolated from the liver. From left to right, FITC labeled XG-102, corresponding DAPI nuclear staining, and merged image of both. In contrast to hepatocytes, which show only background staining levels, FITC staining and cell distribution effectively decorate Kupffer cells. Typical shapes of Kupffer cells and nuclei can be distinguished from hepatocytes by their shape. In contrast to large hexagonal well-organized hepatocytes, Kupffer cells are small triangles. Objective lens 63 times; Bar 20 μm. FIG. 22B shows the localization of FITC-D-TAT (SEQ ID NO: 4) in the liver. It is a figure which shows two cell visual fields of the FITC-D-TAT labeled cell isolated from the liver. From left to right, FITC labeled XG-102, corresponding DAPI nuclear staining, and merged image of both. In contrast to hepatocytes, which show only background staining levels, FITC staining and cell distribution effectively decorate Kupffer cells. Typical shapes of Kupffer cells and nuclei can be distinguished from hepatocytes by their shape. In contrast to large hexagonal well-organized hepatocytes, Kupffer cells are small triangles. Objective lens 63 times; Bar 20 μm. FIG. 23A shows the localization of FITC-XG-102 (SEQ ID NO: 233) in lymph nodes. It is a figure which shows the cell visual field of the FITC-XG-102 labeled cell isolated from the lymph node. The upper panel, from left to right, is composed of one cell field stained with FITC-XG-102 (left), nuclear staining with DAPI (center), and the corresponding merged image (right) It is an image. The lower panel shows an enlarged view of the cell cluster in each image. The cytoplasm of FITC positive cells anatomically staining marginal sinus macrophages and constitutively migrating alveolar macrophages is widely stained with FITC fluorescence. The absence of FITC staining in lymphoid follicles makes it possible to distinguish between myelin cords and cortical areas. Since FITC-XG-102 is incorporated in the medullary region, most of it seems to be composed of macrophages. Objective lens 10 times; Bar 50 μm. FIG. 23B shows the localization of FITC-D-TAT (SEQ ID NO: 4) in lymph nodes. It is a figure which shows the cell visual field of the FITC-D-TAT labeled cell isolated from the lymph node. The upper panel, from left to right, is composed of one cell field stained with FITC-D-TAT (left), nuclear staining with DAPI (center), and the corresponding merged image (right) It is an image. The lower panel shows an enlarged view of the cell cluster in each image. The cytoplasm of FITC positive cells anatomically staining marginal sinus macrophages and constitutively migrating alveolar macrophages is widely stained with FITC fluorescence. The absence of FITC staining in lymphoid follicles makes it possible to distinguish between myelin cords and cortical areas. Since FITC-D-TAT is incorporated in the medullary region, most of it seems to be composed of macrophages. Objective lens 10 times; Bar 50 μm.

  The term “(WB) -targeting (poly) peptide” as used herein can enter leukocytes with high specificity (eg, HepG2 (human hepatocellular carcinoma cells) or HCT-116 cells). (Compared to human colon carcinoma cells) and derived from the HIV TAT protein, ie, the HIV TAT protein (SEQ ID NO: 1; US Pat. Nos. 5,804,604, 5,674,980). Each of these references, including or consisting of an amino acid sequence that is a fragment or variant of (or a variant of such a fragment) (which is incorporated herein by reference) ) Refers to a peptide. Specifically, the fragments, variants, and variants of such fragments are generated from TAT residues 49-57 (SEQ ID NO: 2) or 48-57 (SEQ ID NO: 3). The amino acid sequence may include D-amino acids and / or L-amino acids.

  The term “(poly) peptide” as used herein means a term generally understood in the art, ie, refers to an amino acid chain. However, the terms are not to be construed with a limited amino acid chain length. Moreover, the said chain | strand does not necessarily need to be linear form and may be branched.

  The (poly) peptide targeting the WBC is preferably less than 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 Amino acid residues, more preferably 5 to 150 amino acid residues, most preferably 5 to 100 amino acid residues, particularly preferably 5 to 75 amino acid residues, most preferably 9 to 50 amino acid residues, eg 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 Or contains 50 amino acid residues.

  Examples of sequences of the (poly) peptide targeting WBC may include the sequences shown in Tables 1 to 3, or may be composed of the sequences shown in Tables 1 to 3.

In Tables 1 to 3, D-enantiomer amino acids are shown in lower case letters, and L-enantiomer amino acids are shown in upper case letters. All sequences were read from the N-terminus to the C-terminus (from left to right). “ΒA” refers to β-alanine. In SEQ ID NOs: 6 and 7, each X generally represents an amino acid residue, preferably selected from any (native) amino acid residue. X _n ^a generally represents one amino acid residue, preferably selected from any amino acid residue except serine or threonine, and n (the number of X repeats) is 0 or 1 . Further, each X _n ^b may be selected from any amino acid residue, and n (the number of X repeats) is 0 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 30, or and the more, provided _that when n of _X ^{n a} (number of iterations X) is _{0, X} ^{n _b,} in order to avoid that the position of the _X ^{n a} is serine or threonine, serine at the C-terminus Or it is preferable not to contain threonine. Preferably, X _n ^b represents a continuous (poly) peptide residues derived from SEQ ID NO: 1. X _n ^a and X _n ^b may represent either a D amino acid or an L amino acid.

(Poly) peptides targeting WBC for use in various embodiments of the invention include, for example, the amino acid sequence of HIV TAT residues 49-57 (SEQ ID NO: 2), (chemical) derivatives thereof, or variants thereof Or a (poly) peptide consisting of these, wherein the variant of SEQ ID NO: 2 is
i) a (poly) peptide comprising or consisting of at least one amino acid sequence according to SEQ ID NO: 235, a (chemical) derivative thereof, or a variant thereof; and ii) any one of SEQ ID NOs: 2-116 A (poly) peptide comprising or consisting of at least one such amino acid sequence, a (chemical) derivative thereof, or a variant thereof,
Selected from the group consisting of

In a particularly preferred embodiment, the variant of the HIV TAT protein fragment is a (poly) peptide comprising an amino acid sequence according to any one of SEQ ID NOs: 1-116, more preferably comprising the following amino acid sequence: or it consists of the amino acid sequence is (poly) peptide: L-TAT (SEQ ID NO: 2), D-TAT (SEQ ID NO: _{4), r 3 -L-TAT} ( SEQ ID NO: _15), r 3 -L-TATi (SEQ ID NO: 16), TAT (s2-28) (SEQ ID NO: 48), TAT (s2-64) (SEQ ID NO: 84), or TAT (s2-91) (SEQ ID NO: 111).

In certain embodiments, a (poly) peptide targeting WBC comprises or consists of at least one sequence according to rXXXrXXXr (SEQ ID NO: 235), wherein:
r represents the D-enantiomer arginine;
X is any L-amino acid,
Said X may be selected individually and independently from any other X in SEQ ID NO: 252. Preferably, at least four of the six X (L-amino acids) in SEQ ID NO: 235 are K or R. In another embodiment, the (poly) peptide targeting the WBC according to the invention has the sequence rX ₁ X ₂ X ₃ rX ₄ X ₅ X ₆ r (SEQ ID NO: 235), wherein X ₁ is K , X ₂ is K, X ₃ is R, and X ₄ , X ₅ , and X ₆ are any L-amino acids selected independently of each other) or consist of said sequence. Similarly, the transporter construct according to the present invention has the sequence rX ₁ X ₂ X ₃ rX ₄ X ₅ X ₆ r (SEQ ID NO: 235), wherein X ₄ is Q, X ₅ is R, X ₆ is R, and X ₁ , X ₂ , and X ₃ are any L-amino acids selected independently of each other) or may be composed of the above sequences. In addition, the transporter construct of the present invention has the sequence rX ₁ X ₂ X ₃ rX ₄ X ₅ X ₆ r (SEQ ID NO: 235) (wherein 1, 2, 3, 4, 5, or 6 X amino acid residues are selected from the group consisting of: X ₁ is K, X ₂ is K, X ₃ is R, X ₄ is Q, X ₅ is R , X ₆ is R, while the remaining X amino acid residues not selected from the above group may be any L-amino acid and are selected independently of each other) or consist of said sequence May be. X ₁ is preferably Y and / or X ₄ is preferably K or R. Inverse sequences and / or chemical derivatives of the above sequences, and embodiments of SEQ ID NO: 235 are contemplated as well.

  Furthermore, as long as the (poly) peptide targeting the WBC retains the function / activity of entering the WBC with high specificity, the (poly) peptide targeting the WBC described in Tables 1 to 3 is used. From fragments or variants of Specifically, the variants of the sequences described in Tables 1 to 3 do not have to have the same sequence of D amino acids and L amino acids, that is, they may be composed of all D amino acids, and all of them may be composed of L amino acids. It may be composed of a random mixture of D and L amino acids.

  In the context of the present invention, variants and / or fragments of (poly) peptides have at least 10%, 20%, 30%, 40%, 50% sequence identity over the full length of the (poly) peptide referred to. Comprise or consist of (poly) peptide sequences that are 60%, 70%, 80%, or 85%, preferably at least 90%, more preferably at least 95%, most preferably at least 99% It is preferable. Furthermore, such (poly) peptide fragments may further comprise full-length sequence epitopes (also referred to as “antigenic determinants”). In the context of the present invention, an epitope is generally a fragment located on the outer surface of a (native) protein or (poly) peptide sequence as defined herein, preferably 5 to 15 Of amino acids, more preferably 5 to 12 amino acids, even more preferably 6 to 9 amino acids, and the fragment can be recognized by the antibody, i.e., intact.

  As used herein, the “fragment” of the (poly) peptide targeting WBC specifically disclosed in Tables 1 to 3 is compared to its truncated sequence, ie the amino acid sequence of the original sequence. It is preferably understood that the amino acid sequence is truncated in the N-terminus, C-terminus, and / or sequence.

  Further, in the context of the present invention, “variants” of (poly) peptides are one or more mutations, such as amino acids with one or more substitutions (or insertions and / or deletions, if necessary), etc. Is preferably understood as a sequence in which the amino acid sequence of the variant differs from the sequence of the (poly) peptide (or fragment thereof) referred to. Variants have essentially the same biological function or specific activity compared to the full length original sequence. When referring to the (poly) peptide mutants targeting the WBC, it means that these mutants still transport to WBC cells. Within the above meaning range, the mutant is preferably about 1-50, more preferably 1-20, even more preferably 1-10, particularly preferably 1-5, most preferably Preferably it may contain 4, 3, 2 or 1 amino acid changes. The variant may also contain conservative amino acid substitutions. The conservative amino acid substitution may include substitution of an amino acid residue with another amino acid residue that is sufficiently similar in physicochemical properties so that the biological activity of the molecule is maintained upon substitution. Good (see, eg, Grantham, R. (1974), Science 185, 862-864). Further, the function is changed particularly when the number of amino acids involved in insertion and / or deletion is several, for example, less than 20, preferably less than 10, and amino acids important for functional activity are not removed or displaced. It will be apparent to those skilled in the art that amino acids in the sequences defined above can be inserted and / or deleted without. The conservative amino acid substitution is preferably a substitution for exchanging amino acids derived from the same class of amino acids (basic amino acids, acidic amino acids, polar amino acids, etc.). Related aspects are aliphatic side chains, positively charged side chains, negatively charged side chains, aromatic groups in amino acid side chains, side chains that can provide hydrogen bonding, eg, side chains with hydroxyl functionality. This may, for example, replace an amino acid having a polar side chain with another amino acid that also has a polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain, also having a hydrophobic side chain Substituting with another amino acid (for example, substituting serine (threonine) with threonine (serine) or leucine (isoleucine) with isoleucine (leucine)). Table 4 shows synonymous amino acid residues that are classified in the same group and that are generally interchangeable by conservative amino acid substitutions.

In certain embodiments, the variant is
i) at least one amino acid sequence according to SEQ ID NO: 235, or a (chemical) derivative thereof, or a variant thereof, or a (poly) peptide consisting thereof; and ii) according to any one of SEQ ID NOs: 2-116 A (poly) peptide comprising or consisting of at least one amino acid sequence, a (chemical) derivative thereof, or a variant thereof,
A variant of SEQ ID NO: 2 selected from the group consisting of
The function / activity of a fragment or variant can be tested by various tests such as, for example, transfection efficiency, precise expression of the protein encoded by the cargo nucleic acid, or, for example, spectroscopy, computer modeling, structure It can also be tested by biophysical methods such as analysis. Said function / activity is analyzed by a hydrophilicity analysis (eg Hopp and Woods, 1981. Proc Natl Acad Sci USA 78: 3824-3828) which can be used to identify hydrophobic and hydrophilic regions of (poly) peptides. Thus, it can be used to design the substrate for experimental manipulation. Secondary structure analysis may be performed to identify regions of (poly) peptides or variants and / or fragments thereof to infer specific structural motifs (eg, Chou and Fasman, 1974, Biochem 13: 222-223). Manipulation, translation, secondary structure prediction, hydrophilic and hydrophobic profiles, open reading frame prediction and plotting, and determination of sequence homology can be performed using computer software programs available in the art. Other methods for structural analysis include, for example, X-ray crystal structure analysis (see, eg, Engstrom, 1974. Biochem Exp Biol 11: 7-13), mass spectrometry and gas chromatography (eg, METHODS IN PROTEIN SCIENCE, 1997, J. Wiley and Sons, New York, NY). Computer modeling (e.g. Fletterick and Zoller, eds., 1986. Computer Graphics and Molecular Modeling, In: CURRENT COMMUNICATIONS IN MOLECULAR BIOLOGY, Cold Spring Harbor, Cold Spring Harbor.

  For sequences that do not match exactly (amino acids or nucleic acids), the identity of the first sequencing may be determined taking into account the second sequencing. In general, the two sequences to be compared are aligned so that the correlation between the sequences is maximized. This may include inserting “gaps” in one or both sequences to increase the degree of alignment. Next, the identity (%) relative to the total length of each compared sequence (so-called global alignment), which is particularly suitable for sequences of the same or similar length, or shorter than the full length, which is more suitable for non-equal length sequences The identity (%) (so-called local alignment) to the length of can be determined. In the above situation, for example, an amino acid sequence having “sequence identity” of at least 95% with respect to the query amino acid sequence means that the subject amino acid sequence contains up to 5 amino acid changes for every 100 amino acids in the query amino acid sequence. Except as obtained, it is intended to mean that the sequence of the subject amino acid sequence is identical to the query amino acid sequence. In other words, to obtain an amino acid having a sequence that is at least 95% identical to the query amino acid sequence, preferably within the above definition of the variant or fragment, up to 5% of the amino acid residues in the subject amino acid sequence (5 out of 100) may be inserted, substituted with another amino acid, or deleted.

What has been described above for (poly) peptide variants and fragments applies mutatis mutandis to nucleic acid sequences.

  Methods for comparing the identity and homology of two or more sequences are well known in the art. The rate at which two sequences are identical can be determined, for example, using a mathematical algorithm. The mathematical algorithm is not particularly limited, but Karlin et al. (1993), PNAS USA, 90: 5873-5877. Examples of the algorithm include a BLAST or NBLAST program (Altschul et al., 1990, J. Mol. Biol. 215, 403-410, or Altschul accessible from the NCBI website (www.ncbi.nlm.nih.gov). et al. (1997), Nucleic Acids Res, 25: 3389-3402), and FASTA (Pearson (1990), Methods Enzymol. 183, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. US 85, 2444-2448.) And the like. These programs can identify sequences that are somewhat homologous to other sequences. Further, for example, using a program available in Wisconsin Sequence Analysis Package, Version 9.1 (Devereux et al., 1984, Nucleic Acids Res., 387-395), such as the BESTFIT and GAP programs, between two polynucleotides. Identity (%), identity between two polypeptide sequences (%), and homology or identity. BESTFIT uses the “local homology” algorithm of Smith and Waterman ((1981), J. Mol. Biol. 147, 195-197) to find one region with the highest similarity between two sequences.

  In the context of the present invention, the L-amino acids, also called L-enantiomer amino acids, are preferably amino acids selected from natural amino acids or derivatives thereof. The natural amino acids are generally standard amino acids (constituting proteins) such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, Non-standard amino acids such as ornithine, citrulline, homocysteine, S-adenosylmethionine, hydroxyproline, selenocysteine, pyrrolidine, and lanthionine include phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. , 2-aminoisobutyric acid, dehydroalanine, γ-aminobutyric acid and the like.

  As used herein, the term (chemical) “derivative” refers to a (chemical) modification of an amino acid / amino acid chain at the N-terminus, C-terminus, backbone chain, peptide bond, and / or side chain residue. Point to. The term is not intended to refer to any addition, substitution, or deletion of amino acids in the amino acid chain. Such (chemical) derivatives of L-amino acids or L-enantiomer amino acids generally include, but are not limited to, the amino acids defined above, including but not limited to the following post-translational or synthetic modifications: Any natural or non-natural derivative of an amino acid or L-enantiomer amino acid: acetylation (at the N-terminus of a (poly) peptide sequence, lysine residue, etc.), deacetylation, alkylation, eg methylation Amidation (preferably of (poly) peptide sequence, preferably at lysine or arginine residues within (poly) peptide sequences), dealkylation, eg demethylation, deethylation, etc. Formylation, γ-carboxylation, glutamylation, glycosylation (preferably asparagine within the (poly) peptide sequence), Addition of heme or haem moieties, hydroxylation, iodination, isoprenylation (addition of isoprenoid moieties such as farnesyl or geranylgeraniol), lipoylation (lipoate function) Group), eg, prenylation, GPI anchor formation, eg, myristoylation, farnesylation, geranylgeraniolation, etc. oxidation, phosphorylation (eg, serine, tyrosine, threonine within (poly) peptide sequences) Or histidine moiety), sulfation (eg of tyrosine), selenoylation, sulfation and the like.

The (chemical) derivative of the L-amino acid is not particularly limited, and examples thereof include a modified L-amino acid modified by introducing one of the following labels. As the sign,
(I) a radiolabel, ie, radiophosphorylation, or a radiolabel containing sulfur, hydrogen, carbon, nitrogen, etc .;
(Ii) colored pigments (eg, digoxigenin);
(Iii) a fluorescent group (eg, fluorescein, rhodamine, fluorescent chromoprotein as defined below);
(Iv) a chemiluminescent group;
(V) A combination of two or more of the labels described in (i) to (iv).

  The derivative of the L-amino acid is not particularly limited, and examples thereof include AMC (aminomethylcoumarin), Dabcyl (dimethylaminophenylazobenzoyl), Dansyl (dimethylaminonaphthalenesulfonyl), FAM (carboxyfluorescein), Mca (methoxycoumarin). Acetyl), Xan (xanthyl), Abu (aminobutyric acid), Beta-Ala (β-alanine), E-Ahx (6-aminohexanoic acid), Alpha-Aib (α-aminoisobutyric acid), Ams (aminoserine), Cha (Cyclohexylamine), Dab (diaminobutyric acid), Hse (homoserine), Hyp (hydroxyproline), Mpr (mercaptopropionic acid), Nal (naphthylalanine), Nva (norvaline), Orn (ornithine), Phg (Phenylglycine), Sar (sarcosine), Sec (selenocysteine), Thi (thienylalanine) and the like.

  Furthermore, the L-enantiomer amino acid selected may be selected from a specific combination of the L-enantiomer amino acids defined above or derivatives thereof. Such combinations include one, two, three, four, five, six, seven, eight, nine, 10 of the L-enantiomer amino acids or derivatives thereof defined above. Individual or more may be included. Combinations of any of the L-enantiomer amino acids or derivatives thereof defined above with any of the D-enantiomer amino acids or derivatives thereof defined in more detail below are also possible. Certain combinations of such amino acids may exhibit higher or lower stability against peptidases, for example, increasing the in vivo or in vitro stability of (poly) peptides targeting WBC, or A further possibility of lowering can be provided. For example, (poly) peptides that target WBC are D and / or L (ie, D-enantiomer amino acids, L-enantiomer amino acids, or D-enantiomer amino acids and L-enantiomers). (Mixture with amino acids) preferably may contain the L-shaped dipeptide sequence Arg-Lys, but this is less stable against peptidases, so that (poly) peptides of (poly) peptides targeting WBC The sequence can be destabilized and used to further reduce in vivo half-life.

  In the context of the present invention, the D-amino acid, also referred to as the D-enantiomer amino acid, is preferably a non-native (does not constitute a protein) amino acid, and these non-native (does not constitute a protein) It is preferably derived from natural L-amino acids and / or derivatives thereof as defined in. “D-amino acid” refers to an isomer of a natural L-amino acid as defined above (and a (poly) peptide made therefrom) in which the chirality of the natural L-amino acid residue is the inverse of the corresponding D-amino acid. (See, eg, Jameson et al., Nature, 368, 744-746 (1994); Brady et al., Nature, 368, 692-693 (1994)). In other words, in the (poly) peptide bond of D-amino acid, the position of the carbonyl group and the amino group are interchanged, but the position of the side chain group in each α carbon is conserved. Thus, the D-amino acid can be inserted into a (poly) peptide sequence that consists of or comprises the L-amino acid, and is defined above by methods known in the art. Can be conjugated with L-amino acids. Known methods in the art include, for example, a liquid phase (poly) peptide synthesis method or a solid (poly) peptide synthesis method, such as a solid (poly) peptide synthesis method by Merrifield, a t-Boc solid phase (poly) peptide. Synthesis, Fmoc solid phase (poly) peptide synthesis, solid phase (poly) peptide synthesis based on BOP (benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate), etc. It is not limited to. The content of the D-amino acid further provides various useful properties. For example, such (poly) peptides are more stable and less immunogenic (especially in vivo) than (poly) peptides that target WBC based on the corresponding L-amino acid sequence. However, in particular, due to the fact that almost all degrading enzymes such as proteases or peptidases cleave (poly) peptide bonds between adjacent L-amino acids, the polypeptides can alleviate WBCs made of all D-amino acids. It does not remain in the cell as much as the targeted (poly) peptide. As a result, (poly) peptides consisting of D-enantiomeric amino acids and L-enantiomeric amino acids are resistant to rapid proteolysis that does not lead to accumulation in cells because they are free from protease degradation. .

  The (poly) peptide targeting WBC preferably comprises the L-amino acids and D-amino acids defined above or derivatives thereof. Such derivatives are present in the entire (poly) peptide targeting WBC in about 0%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about It may be contained in a content of 80%, about 90%, or even about 100%. In other words, the entire (poly) peptide targeting WBC has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 such derivatives. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more You may contain above.

  A (poly) peptide targeting WBC for use in the present invention does not consist of the following sequences: KRIIQRILSRNS (SEQ ID NO: 236); KRIHPRLTRSIR (SEQ ID NO: 237); PPRLRKRRQLNM (SEQ ID NO: 238); PIRRRKKLRRRLK (sequence) RRQRRTSKLMKR (SEQ ID NO: 240); MHKRPTTSPSRKM (SEQ ID NO: 241); RQRSRRRPPLNIR (SEQ ID NO: 242); RIRMIQNLIKKT (SEQ ID NO: 243); 246); HRHIRRQSLIML (SEQ ID NO: 247); PQNRLQIRRHSK (SEQ ID NO: 248); PP SNRQRNLNM (SEQ ID NO: 249); SMLKRNHSTSNR (SEQ ID NO: 250); GSHPPSLIIPRQ (SEQ ID NO: 251); SPMQKTMNLPPM (SEQ ID NO: 252); NKRILIRIMTRP (SEQ ID NO: 253); (SEQ ID NO: 256); LTRNYEAWVPTP (SEQ ID NO: 257); SAETVESCLAKSH (SEQ ID NO: 258); YSHIATLTPFTPT (SEQ ID NO: 259); SEQ ID NO: 263); HFAAWG HSLVH (SEQ ID NO: 264); HIQLSFSFSQSWR (SEQ ID NO: 265); LTMPSDLQPVLW (SEQ ID NO: 266); FQPYPDHPAEVSY (SEQ ID NO: 267); (SEQ ID NO: 271); SLGWMLFPSPPF (SEQ ID NO: 272); SHAFTWPTYLQL (SEQ ID NO: 273); SHNWLPLWPLRP (SEQ ID NO: 274); SEQ ID NO: 278); LNVPPPSWFLS LDITPFLSLLP (SEQ ID NO: 280); LPHPVLHMGPLR (SEQ ID NO: 281); VSKQPYYMWNGN (SEQ ID NO: 282); NYTTYKSHQQDR (SEQ ID NO: 283); (SEQ ID NO: 286); DPATNPGPHFPR (SEQ ID NO: 287); TLSPPLALLTVH (SEQ ID NO: 288); HPGSFPFPPEHRP (SEQ ID NO: 289); TSHTDAPPPARSP (SEQ ID NO: 290); MTPSSLSTLPWP (SEQ ID NO: 291); SEQ ID NO: 293); TPKTMTQTYDFS ( NSGTMQSASRAT (SEQ ID NO: 295); QAASRVENYMHR (SEQ ID NO: 296); HQHKPPPLTNNN (SEQ ID NO: 297); SNPWDSLLSVST (SEQ ID NO: 298); GVVGKLGQRRTKKQRRQKK (SEQ ID NO: 302); GRRTKKQRRQKKPPRYMILGLLALAAVCSAA (SEQ ID NO: 303); GRRTKKQRRQKKPP (SEQ ID NO: 304). In certain embodiments, the (poly) peptide targeting WBC for use in embodiments of the present invention does not comprise or consist of SEQ ID NO: 1.

  The object underlying the present invention is solved by binding one, two or more (poly) peptides targeting the WBC to at least one further substance (load). The combination of (poly) peptide and cargo that targets WBC is hereinafter referred to as "transporter-load conjugate molecule". The present invention therefore also comprises a transporter comprising as component (A) at least one WBC according to the present invention (poly) peptide and a further substance (load) as component (B) -Relates to cargo conjugate molecules. Of course, the transporter-loading conjugate molecule may include any number of additional components (C), (D), (E), and the like. In the following, component (B) will be described in more detail, but these features can also be applied to any other additional components (C), (D), (E), etc.

  Those skilled in the art will appreciate that the inventors' contribution to the art is primarily to provide a general means by which the cargo can be efficiently delivered to the leukocytes. Thus, the actual component (B) of the transporter-loading conjugate molecule according to the present invention may in principle be any substance that one skilled in the art wants to introduce into the leukocytes for any reason. Such reasons include, but are not limited to, therapeutic reasons (such as treatment, prevention, attenuation or recovery of the disease), diagnostic reasons, scientific reasons, technical reasons, commercial reasons, etc. Including. Some examples of the component (B) are shown below. However, the above examples are not to be construed as limiting the scope of the invention.

For example, the cargo molecule (component (B)) can be selected from at least one of the following:
a) proteins and / or (poly) peptides comprising therapeutically active proteins and / or (poly) peptides;
b) a protein kinase inhibitor comprising an inhibitor of c-Jun amino terminal kinase or a factor that is a protein kinase;
c) an antigen,
d) antibodies,
e) an apoptotic factor,
f) a protease involved in a medical condition, comprising a peptide protease inhibitor;
g) BH3-domain,
h) BH3-only protein,
i) DNA,
j) RNA, including siRNA, antisense RNA, microRNA,
k) cytotoxic agents,
l) small organic compounds,
m) small molecule drugs,
n) gold particles,
o) fluorescent dyes,
p) antibiotics, and q) static virus agents.

  In the context of the present invention, therapeutically active proteins or (poly) peptides suitable as effector molecules for component (B) of the transporter-loading conjugate molecules of the present invention are intracellular signaling such as cytokines, antibodies, etc. Can be selected from, but not limited to, proteins that can be stimulated or inhibited. Thus, the therapeutically active protein has four cysteine residues (CCCC) that are conserved in position and the conserved sequence motif Trp-Ser-X-Trp-Ser (WSXWS), where X is conserved. May include a class I cytokine family of cytokines. The cytokines of the class I cytokine family include, for example, GM-CSF subfamily such as IL-3, IL-5, GM-CSF, for example, IL-6 subfamily such as IL-6, IL-11, IL-12, For example, IL-2, IL-4, IL-7, IL-9, IL-2 subfamily such as IL-15, or cytokines IL-1α, IL-1β, IL-10 and the like are included. A therapeutically active protein also has a class II cytokine family of cytokines that have four cysteine residues (CCCC) that are conserved in position but do not contain the conserved sequence motif Trp-Ser-X-Trp-Ser (WSXWS). May be included. Class II cytokine family cytokines include, for example, IFN-α, IFN-β, IFN-γ, and the like. The therapeutically active proteins include, for example, cytokines of the family of tumor necrosis factors such as TNF-α, TNF-β, for example, seven transmembrane helices such as IL-8, MIP-1, RANTES, CCR5, CXR4, And a chemokine family cytokine that interacts with G-protein, for example, a cytokine-specific receptor such as TNF-RI, TNF-RII, CD40, OX40 (CD134), Fas, or fragments or variants thereof. Good. Such fragments and variants preferably exhibit sequence homology or sequence identity as defined above.

The therapeutically active protein suitable as component (B) of the transporter-loading conjugate molecule according to the present invention may also be selected from any protein shown in the following non-exhaustive list: 0ATL3, 0FC3, 0PA3 , 0PD2, 4-1BBL, 5T4, 6Ckine, 707-AP, 9D7, A2M, AA, AAAS, AACT, AASS, ABAT, ABCA1, ABCA4, ABCB1, ABCB11, ABCB2, ABCB4, ABCB7, ABCB1, BCC7 , ABCD3, ABCG5, ABCG8, ABL1, ABO, ABR ACAA1, ACACA, ACADL, ACADM, ACADS, ACADVL, ACAT1, ACCPN, ACE, ACHE, ACHM3, ACHM1, ACLS, ACPI, A CTA1, ACTC, ACTN4, ACVRL1, AD2, ADA, ADAMTS13, ADAMTS2, ADFN, ADH1B, ADH1C, ALDDH3A2, ADRB2, ADRB3, ADSL, AEZ, AFA, AFD1, AFP, AGA, AGL, AGMX1, AGT, AGMX1 AGTR1, AGXT, AH02, AHCY, AHDS, AHHR, AHSG, AIC, AIED, AIH2, AIH3, AIM-2, AIPL1, AIRE, AK1, ALAD, ALAS2, ALB, HPG1, ALDH2, ALDH3A1, ALDH4A1, ALDH4A1, ALDH4A1 ALDOA, ALDOB, ALMS1, ALPL, ALPP, ALS2, ALX4, AMACR, AMBP, AMCD, AMCD1, AMC , AMLEL, AMLEY, AMGL, AMH, AMHR2, AMPD3, AMPD1, AMT, ANC, ANCR, ANK1, ANOP1, AOM, AP0A4, AP0C2, AP0C3, AP3B1, APC, aPKC, APOA2, APOA1, APOB, POOC3, POPO, APOC3 , APOH, APP, APRT, APS1, AQP2, AR, ARAF1, ARG1, ARGGEF12, ARMET, ARSA, ARSB, ARSC2, ARSE, ART-4, ARTTC1 / m, ARTS, ARVD1, ARX, AS, ASAH, ASAT, ASD1 , ASL, ASMD, ASMT, ASNS, ASPA, ASS, ASSP2, ASSP5, ASSP6, AT3, ATD, ATHS, ATM, ATP2A1, ATP2A2 ATP2C1, ATP6B1, ATP7A, ATP7B, ATP8B1, ATPSK2, ATRX, ATXN1, ATXN2, ATXN3, AUTS1, AVMD, AVP, AVPR2, AVSD1, AXIN1, AXIN2, AZF2, B2AG, B4GHT7, B4GALT7 BBS2, BBS3, BBS4, BCA225, BCAA, BCH, BCHE, BCKDHA, BCKDHB, BCL10, BCL2, BCL3, BCL5, BCL6, BCPM, BCR, BCR / ABL, BDC, BDE, BDMF, BDMR, BEST / β-catenin m, BF, BFHD, BFIC, BFLS, BFSP2, BGLAP, BGN, BHD, BHR1, BING-4, BIRC5, BJS, BLM, BLM , BLNK, BMPR2, BPGM, BRAF, BRCA1, BRCA1 / m, BRCA2, BRCA2 / m, BRCD2, BRCD1, BRDT, BSCL, BSCL2, BTAA, BTD, BTK, BUB1, BWS, BZX, C0L2A1, C0L2A1, C0L2A1, C0L2A1 , C1QB, C1QG, C1S, C2, C3, C4A, C4B, C5, C6, C7, C7orf2, C8A, C8B, C9, CA125, CA15-3 / CA27-29, CA195, CA19-9, CA72-4, CA2 CA242, CA50, CABYR, CACD, CACNA2D1, CACNA1A, CACNA1F, CACNA1S, CACNB2, CACNB4, CAGE, CA1, CALB3, CALCA, CALCR, CALM, CAL , CAM43, CAMEL, CAP-1, CAPN3, CARD15, CASP-5 / m, CASP-8, CASP-8 / m, CASR, CAT, CATM, CAV3, CB1, CBBM, CBS, CCA1, CCAL2, CCAL1, CCAT , CCL-1, CCL-11, CCL-12, CCL-13, CCL-14, CCL-15, CCL-16, CCL-17, CCL-18, CCL-19, CCL-2, CCL-20, CCL -21, CCL-22, CCL-23, CCL-24, CCL-25, CCL-27, CCL-3, CCL-4, CCL-5, CCL-7, CCL-8, CCM1, CCNB1, CCND1, CCO , CCR2, CCR5, CCT, CCV, CCZS, CD1, CD19, CD20, CD22, CD25, CD27, CD27L, cD3, CD30, CD30, CD30L, CD33, CD36, CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD44v, CD44v6, CD52, CD55, CD56, CD59, CD80, CD86, CDAN1, CDAN2, CDAN3, CDC27, CDC27 / m, CDC2L1, CDH1, CDK4, CDK4 / m, CDKN1C, CDKN2A, CDKN2A / m, CDKN1A, CDKN1C, CDL1, CDPD1, CDR1, CEA, CEACAM1, CTPAMC1, EC9C CFNS, CFTR, CGF1, CHAC, CHED2, CHED1, CHEK2, CHM, CHML, CHR39C, CHRNA4, CHR A1, CHRNB1, CHRNE, CHS, CHS1, CHST6, CHX10, CIAS1, CIDX, CKN1, CLA2, CLA3, CLA1, CLCA2, CLCN1, CLCN5, CLCNKB, CLDN16, CLP, CLN2, CLN5, CLN4, CLN4, CLN4, CLN4 C1QA, C1QB, C1QG, C1R, CLS, CMCWTD, CMDJ, CMD1A, CMD1B, CMH2, MH3, CMH6, CMKBR2, CMKBR5, CML28, CML66, CMM, CMT2B, CMT2C, CMT4A, CMT1C CNA1, CND, CNGA3, CNGA1, CNGB3, CNSN, CNTF, COA-1 / m, COCH, COD2, COD1, COH1, OL10A, COL2A2, COL11A2, COL17A1, COL1A1, COL1A2, COL2A1, COL3A1, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL7A1, COL8A2, COL9A2, COL9A3, COL11A1, COL1A2, COL23A1, COL1A1, COLQ, COMP, COMT, CORD5, CORD1, COX10, COX-2, CP, CPB2, CPO, CPP, CPS1, CPT2, CPT1A, CPX, CRAT, CRB1, CRBM, CREBBP, CRH, CRHBP, CRS, CRV, CRX, CRYAB, CRYBA1, CRYBB2, CRYGA, CRYGC, CRYG D, CSA, CSE, CSF1R, CSF2RA, CSF2RB, CSF3R, CSF1R, CST3, CSTB, CT, CT7, CT-9 / BRD6, CTAA1, CTACK, CTEN, CTH, CTHM, CTLA4, CTM, CTNNB1, CTNS, CTPA, CTSB, CTSC, CTSK, CTSL, CTS1, CUBN, CVD1, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL7, CXCL8 CYBB5, CYFRA21-1, CYLD, CYLD1, CYMD, CYP11B1, CYP11B2, CYP17, CYP17A1, CYP 9, CYP19A1, CYP1A2, CYP1B1, CYP21A2, CYP27A1, CYP27B1, CYP2A6, CYP2C, CYP2C19, CYP2C9, CYP2D, CYP2D6, CYP2D7P1, CYP3A4, CYP7B1, CYPB1, CYP11B1, CYP1A1, CYP1B1, CYRAA, D40, DADl, DAM, DAM- 10 / MAGE-B1, DAM-6 / MAGE-B2, DAX1, DAZ, DBA, DBH, DBI, DBT, DCC, DC-CK1, DCK, DCR, DCX, DDB1, DDB2, DDIT3, DDU, DECR1, DEK- CAN, DEM, DES, DF, DFN2, DFN4, DFN6, DFNA4, DFNA5, DFNB5, DGCR, DHCR7, DHFR, DH F, DHS, DIA1, DIAPH2, DIAPH1, DIH1, DIO1, DISCI, DKC1, DLAT, DLD, DLL3, DLX3, DMBT1, DMD, DM1, DMPK, DMWD, DNAI1, DNASE1, DNMT3B, DPEP1, DPYD, DPYS DRD4, DRPLA, DSCR1, DSG1, DSP, DSPP, DSS, DTDP2, DTR, DURS1, DWS, DYS, DYSF, DYT2, DYT3, DYT4, DYT2, DYT1, DYX1, EBAF, EBM, EBNA, EBP, EBNA, EBP ECA1, ECB2, ECE1, ECGF1, ECT, ED2, ED4, EDA, EDAR, ECA1, EDN3, EDNRB, EEC1, EEF1A1L14, EEGV1, EF EMP1, EFTUD2 / m, EGFR, EGFR / Her1, EGI, EGR2, EIF2AK3, eIF4G, EKV, El IS, ELA2, ELF2, ELF2M, ELK1, ELN, ELONG, EMD, EML1, ENML2, EMX2, AMX , END3, ENG, ENO1, ENPP1, ENUR2, ENUR1, EOS, EP300, EPB41, EPB42, EPCAM, EPD, EphA1, EphA2, EphA3, Ephrin A2, Ephrin A3, EPHX1, EPM2A, EPO, EPOR, EPX, ERB2, , ERCC3, ERCC4, ERCC5, ERCC6, ERVR, ESR1, ETFA, ETFB, ETFDH, ETM1, ETV6-AML1, ETV1, EVC, EVR2, EVR1, EWSR1, EXT2, EXT3, EXT1, EYA1, EYCL2, EYCL3, EYCL1, EZH2, F10, F11, F12, F13A1, F13B, F2, F5, F5F8D, F7, F8, FA8P, F8, F8C FAH, FANCA, FANCB, FANCC, FANCD2, FANCF, FasL, FBN2, FBN1, FBP1, FCG3RA, FCGR2A, FCGR2B, FCGR3A, FCHL, FCMD, FCP1, FDPSL5, FECH, FEO1, GEF1F FGF2, FGF23, FGF5, FGFR2, FGFR3, FGFR1, FGG, FGS1, FH, FIC1, FIH, F2, FKBP6, FLNA, FLT4, FMO , FMO4, FMR2, FMR1, FN, FN1 / m, FOXC1, FOXE1, FOXL2, FOXO1A, FPDMM, FPF, Fra-1, FRAXF, FRDA, FSHB, FSHMD1A, FSHR, FTH1, FTHL1, FTHL17T , FUT6, FUT1, FY, G250, G250 / CAIX, G6PC, G6PD, G6PT1, G6PT2, GAA, GABRA3, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE -7b, GAGE-8, GALC, GALE, GALK1, GALNS, GALT, GAMT, GAN, GAST, GASTRIN17, GATA3, GATA, GBA, GBE, GC, GCDH, GCGR, GCH , GCK, GCP-2, GCS1, G-CSF, GCSH, GCSL, GCY, GDEP, GDF5, GDI1, GDNF, GDXY, GFAP, GFND, GGCX, GGT1, GH2, GH1, GHR, GHRHR, GHS, GIF, GINGF , GIP, G
JA3, GJA8, GJB2, GJB3, GJB6, GJB1, GK, GLA, GLB, GLB1, GLC3B, GLC1B, GLC1C, GLDC, GLI3, GLP1, GLRA1, GLUD1, GM1 (fuc-GM1, G2) , GNAI2, GNAS, GNAT1, GNB3, GNE, GNPTA, GNRH, GNRH1, GNRHR, GNS, GnT-V, gp100, GP1BA, GP1BB, GP9, GPC3, GPD2, GPDS1, GPI, GP1BA, GP1L, GP1L, GP1L , GPX1, GRHPR, GRK1, GROα, GROβ, GROγ, GRPR, GSE, GSM1, GSN, GSR, GSS, GTD, GTS, GUCA1A, GUCY2D, GULOP, GU B, GUSM, GUST, GYPA, GYPC, GYS1, GYS2, H0KPP2, H0MG2, HADHA, HADHB, HAGE, HAGH, HAL, HAST-2, HB1, HBA2, HBA1, HBB, HBBP1, HBD, HBE1, HBD, HBE HBHR, HBP1, HBQ1, HBZ, HBZP, HCA, HCC-1, HCC-4, HCF2, HCG, HCL2, HCL1, HCR, HCVS, HD, HPN, HER2, HER2 / NEU, HER3, HERV-K-MEL, HESX1, HEXA, HEXB, HF1, HFE, HF1, HGD, HHC2, HHC3, HHG, HK1 HLA-A, HLA-A ^* 0201-R170I, HLA-A11 / m, HLA-A2 / m, HLA-DPB1 HLA- DR , HLCS, HLXB9, HMBS, HMGA2, HMGCL, HMI, HMN2, HMOX1, HMS1 HMW-MAA, HND, HNE, HNF4A, HOAC, HOMEOBOX NKX 3.1, HOM-TES-14 / SCP-STE-HOM, 85, HOXA1 HOXD13, HP, HPC1, HPD, HPE2, HPE1, HPFH, HPFH2, HPRT1, HPS1, HPT, HPV-E6, HPV-E7, HR, HRAS, HRD, HRG, HRPT2, HRP1, HRX11, BHR , HSD17B4, HSD3B2, HSD3B3, HSN1, HSP70-2M, HSPG2, HST-2, HTC2, HTC1, hTERT, HTN3, HTR2C, HVBS6, HVBS1, HVEC HV1S, HYAL1, HYR, I-309, IAB, IBGC1, IBM2, ICAM1, ICAM3, iCE, ICHQ, ICR5, ICR1, ICS1, IDDM2, IDDM1, IDS, IDUA, IF, IFNa / b, IFNGR1, IGAD1, IGER IGF-1R, IGF2R, IGF1, IGH, IGHC, IGHG2, IGHG1, IGHM, IGHR, IGKC, IHG1, IHH, IKBKG, IL1, IL-1 RA, IL10, IL-11, IL12, IL12RB1, IL13, IL-13Rα2 IL-15, IL-16, IL-17, IL18, IL-1a, IL-1α, IL-1b, IL-1β, IL1RAPL1, IL2, IL24, IL-2R, IL2RA, IL2RG, IL3, IL3RA, I 4, IL4R, IL4R, IL-5, IL6, IL-7, IL7R, IL-8, IL-9, immature laminin receptor, IMMP2L, INDX, INFGR1, INFGR2, INFα, IFN, INFγ, INS, INSR, INVS, IP-10, IP2, IPF1, IP1, IRF6, IRS1, ISCW, ITGA2, ITGA2B, ITGA6, ITGA7, ITGB2, ITGB3, ITGB4, ITIH1, ITM2B, IV, IVD, JAG1, JAK3, JBS, JBTS1, JMS JPD, KAL1, KAL2, KALI, KLK2, KLK4, KCNA1, KCNE2, KCNE1, KCNH2, KCNJ1, KCNJ2, KCNJ1, KCNQ2, KCNQ3, KCNQ4, KCNQ1, KCS, KERA KFS, KFSD, KHK, ki-67, KIAA0020, KIAA0205, KIAA0205 / m, KIF1B, KIT, KK-LC-1, KLK3, KLKB1, KM-HN-1, KMS, KNG, KNO, K-RAS / m, KRAS2, KREV1, KRT1, KRT10, KRT12, KRT13, KRT14, KRT14L1, KRT14L2, KRT14L3, KRT16, KRT16L1, KRT16L2, KRT17, KRT18, KRT2A, KRT3, KRT4, KRT5, KRT6 H, KRT6 H , KSA, KSS, KWE, KYNU, L0H19CR1, L1CAM, LAGE, LAGE-1, LALL, LAMA2, LAMA3, LAMB3, LAMB , LAMC2, LAMP2, LAP, LCA5, LCAT, LCCS, LCCS1, LCFS2, LCS1, LCT, LDHA, LDHB, LDHC, LDLR, LDLR / FUT, LEP, LEWISY, LGCR, LGGF-PBP, LGI1, LGD1H, LGD1 , LHB, LHCGR, LHON, LHRH, LHX3, LIF, LIG1, LIMM, LIMP2, LIPA, LIPA, LIPB, LIPC, LIVIN, L1CAM, LMAN1, LMNA, LMX1B, LOLR, LOR, LOX, PPL, LPL, LPL , LRP5, LRS 1, LSFC, LT-β, LTBP2, LTC4S, LYL1, XCL1, LYZ, M344, MA50, MAA, MADH4, MAFD2, MAF 1, MAGE, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE1, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE- E2, MAGE-F1, MAGE-H1, MAGE2, MGB1, MGB2, MAN2A1, MAN2B1, MANBA, MANBB, MAOA, MAOB, MAPK8IP1, MAPT, MART-1, MART-2, MART2 / m, MAT1A, MBL2, MBP MB S1, MC1R, MC2R, MC4R, MCC, MCCC2, MCCC1, MCDR1, MCF2, MCKD, MCL1, MC1R, MCOLN1, MCOP, MCOR, MCP-1, MCP-2, MCP-3, MCP-4, MCPH2, MCPH1, MCS, M-CSF, MDB, MDCR, MDM2, MDRV, MDS 1, ME1, ME1 / m, ME2, ME20, ME3, MEAX, MEB, MEC CCL-28, MECP2, MEFV, MELANA, MELAS, MEN1 MSLN, MET , MF4, MG50, MG50 / PXDN, MGAT2, MGAT5, MGC1 MGCR, MGCT, MGI, MGP, MHC2TA, MHS2, MHS4, MIC2, MIC5, MIDI, MIF, MIP, MIP-5 / HCC-2, M ITF, MJD, MKI67, MKKS, MKS1, MLH1, MLL, MLLT2, MLLT3, MLLT7, MLLT1, MLS, MLYCD, MMA1a, MMP11, MMVP1, MN / CA IX-antigen, MNG1, MN1, MOC31 MOG, MORC, MOS, MOV18, MPD1, MPE, MPFD, MPI, MPIF-1, MPL, MPO, MPS3C, MPZ, MRE11A, MROS, MRP1, MRP2, MRP3, MRSD, MRX14, MRX2, MRX20, MRX3, MRX40, MRXA, MRX1, MS, MS4A2, MSD, MSH2, MSH3, MSH6, MSS, MSSE, MSX2, MSX1, MTATP6, MTC03, MTCO1, MTCYB, MTHFR MTM1, MTMR2, MTND2, MTND4, MTND5, MTND6, MTND1, MTP, MTR, MTRNR2, MTRNR1, MTRR, MTTE, MTTG, MTTI, MTTK, MTTL2, MTTL1, MTTN, MTTP, MTTS1, MUC1, MUC1, MUC1 MUM-1, MUM-1 / m, MUM-2, MUM-2 / m, MUM-3, MUM-3 / m, MUT, mutant p21ras, MUTYH, MVK, MX2, MXI1, MY05A, MYB, MYBPC3 , MYC, MYCL2, MYH6, MYH7, MYL2, MYL3, MYMY, MYO15A, MYO1G, MYO5A, MYO7A, MYOC, myosin / m, MYP2, MYP1, NA88-A, N-acetylglucosamyl Transferase-V, NAGA, NAGLU, NAMSD, NAPB, NAT2, NAT, NBIA1, NBS1, NCAM, NCF2, NCF1, NDN, NDP, NDUFS4, NDUFS7, NDUFS8, NDUVV1, NUFV1, NEB, NEF, NEB, NEF neo-PAP / m, NEU1, NEUROD1, NF2, NF1, NFYC / m, NGEP, NHS, NKS1, NKX2E, NM, NME1, NMP22, NMTC, NODAL, NOG, NOS3, NOTCH3, NOTCH1, NP, NPC2, NPC1, NPC2 NPHL2, NPHP1, NPHS2, NPHS1, NPM / ALK, NPPA, NQO1, NR2E3, NR3C1, NR3C2, NRAS, NRAS / m, NRL, NROB , NRTN, NSE, NSX, NTRK1, NUMA1, NXF2, NY-CO1, NY-ESO1, NY-ESO-B, NY-LU-12, ALDOA, NYS2, NYS4, NY-SAR-35, NYS1, NYX, OA3 , OA1, OAP, OASD, OAT, OCA1, OCA2, OCD1, OCRL, OCRL1, OCT, ODDD, ODT1, OFC1, OFD1, OGDH, OGT, OGT / m, OPA2, OPA1, OPD1, OPEM, OPG, OPN, OPN1L , OPN1MW, OPN1SW, OPPG, OPTB1, TTD, ORM1, ORP1, OS-9, OS-9 / m, OSM LIF, OTC, OTOF, OTSC1, OXCT1, OYTES1, P15, P190 MINOR BCR-ABL, P2RY 2, P3, P16, P40, P4HB, P-501, P53, P53 / m, P97, PABPN1, PAFAH1B1, PAFAH1P1, PAGE-4, PAGE-5, PAH, PAI-1, PAI-2, PAK3, PAP, PAPPA, PARK2, PART-1, PATE, PAX2, PAX3, PAX6, PAX7, PAX8, PAX9, PBCA, PBCRA1, PBT, PBX1, PBXP1, PC, PCBD, PCCA, PCCB, PCK2, PCK1, PCLD, PCOS1, PCSK1 PDB1, PDCN, PDE6A, PDE6B, PDEF, PDGFB, PDGFR, PDGFRL, PDHA1, PDR, PDX1, PECAM1, PEE1, PEO1, PEPD, PEX10, PEX12, PEX13, PEX3 , PEX5, PEX6, PEX7, PEX1, PF4, PFBI, PFC, PFKFB1, PFKM, PGAM2, PGD, PGK1, PGK1P1, PGL2, PGR, PGS, PHA2A, PHB, PHEX, PHGDH, PHKA2, GPH2, PHKAP , PHYH, PI, PI3, PIGA, PIM1-KINASE, PIN1, PIP5K1B, PITX2, PITX3, PKD2, PKD3, PKD1, PKDTS, PKHD1, PKLR, PKP1, PKU1, PLA2G1A, PLA2PL7, PL2PL7, PL2PL7 , PLP1, PMEL17, PML, PML / RARα, PMM2, PMP22, PMS2, PMS1, PNKD, PNLIP, POF1, POLA, PO H, POMC, PON2, PON1, PORC, POTE, POU1F1, POU3F4, POU4F3, POU1F1, PPAC, PPARG, PPCD, PPGB, PPH1, PPKB, PPMX, PPOX, PPP1
R3A, PPP2R2B, PPT1, PRAME, PRB, PRB3, PRCA1, PRCC, PRD, PRDX5 / m, PRF1, PRG4, PRKAR1A, PRKCA, PRKDC, PRKWNK4, PRNP, PROC, PRODH, PROM1, PROP1, PROST8 PRPF31, PRPF8, PRPH2, PRPS2, PRPS1, PRS, PRSS7, PRSS1, PRTN3, PRX, PSA, PSAP, PSCA, PSEN2, PSEN1, PSG1, PSGR, PSM, PSMA, PSORS1, PTC, PTCH, PTCH1, PTCH, PTEN, PTGS1, PTH, PTHR1, PTLAH, PTOS1, PTPN12, PTPNI1, PTPRK, PTPRK / m, PTS, P UJO, PVR, PVRL1, PWCR, PXE, PXMP3, PXR1, PYGL, PYGM, QDPR, RAB27A, RAD54B, RAD54L, RAG2, RAGE, RAGE-1, RAG1, RAP1, RARA, RASA1, RBAF600 / R, RBAF600 / R, R4 RBP4, RBS, RCA1, RCAS1, RCCP1, RCD1, RCV1, RDH5, RDPA, RDS, RECQL2, RECQL3, RECQL4, REG1A, REHOBE, REN, RENBP, RENS1, RET, RFX5, RFXANK, RFX CD168, RHD, RHO, Rip-1, RLBP1, RLN2, RLN1, RLS, RMD1, RMRP, ROM1, ROR2, RP, RP1, RP 4, RP17, RP2, RP6, RP9, RPD1, RPE65, RPGR, RPGRIP1, RP1, RP10, RPS19, RPS2, RPS4X, RPS4Y, RPS6KA3, RRAS2, RS1, RSN, RSS, RU1, RU2, RUNX1, RWNX RYR1, S-100, SAA1, SACS, SAG, SAGE, SALL1, SARDH, SART1, SART2, SART3, SAS, SAX1, SCA2, SCA4, SCA5, SCA7, SCA8, SCA1, SCC, SCCD, SCF, SCLC1, SCN1A, SCN1B, SCN4A, SCN5A, SCNN1A, SCNN1B, SCNN1G, SCO2, SCP1, SCZD2, SCZD3, SCZD4, SCZD6, SCZD1, SDF- a / SDHA, SDHD, SDYS, SEDL, SERPENA7, SERPINA3, SERPINA6, SERPINA1, SERPINC1, SERPIND1, SERPINE1, SERPINF2, SERPING1, SERPINI1, SFTPPA1, SGTPSG, SGPCD, SGTPD, SGPC SGY-1, SH2D1A, SHBG, SHFM2, SHFM3, SHFM1, SHH, SHOX, SI, SIAL, SIALY LEWISX, SIASD, S11, SIM1, SIRT2 / m, SIX3, SJS1, SKP2, LC1A12S, LC1A19S , SLC22A1L, SLC22A5, SLC25 13, SLC25A15, SLC25A20, SLC25A4, SLC25A5, SLC25A6, SLC26A2, SLC26A3, SLC26A4, SLC2A1, SLC2A2, SLC2A4, SLC3A1, SLC4A1, SLC4A4, SLC3A5, SLC5A1, SLC6A5 SMAD1, SMAL, SMARCB1, SMAX2, SMCR, SMCY, SM1, SMN2, SMN1, SMPD1, SNCA, SNRPN, SOD2, SOD3, SOD1, SOS1, SOST, SOX9, SOX10, Sp17, SPANXC, SPG23, SPG3PG, SPG4, SPG3PG SPG5B, SPG6, SPG7, SP NK1, SPINK5, SPPK, SPPM, SPSMA, SPTA1, SPTB, SPTLC1, SRC, SRD5A2, SRPX, SRS, SRY, βhCG, SSTR2, SSX1, SSX2 (HOM-MEL-40 / SSX2), SSX4, ST8, STAMP-1 , STAR, STARP, STATH, STEAP, STK2, STK11, STn / KLH, STO, STOM, STS, SUOX, SURF1, SURVIVIN-2B, SYCP1, SYM1, SYN1, SYNS1, SYP, SYT / SSX, SYT / SSX, SYT , SYT-SSX-2, TA-90, TAAL6, TACSTD1, TACSTD2, TAG72, TAF7L, TAF1, TAGE, TAG-72, TALI, TAM, TAP2, TAP1, APVR1, TARC, TARP, TAT, TAZ, TBP, TBX22, TBX3, TBX5, TBXA2R, TBXAS1, TCAP, TCF2, TCF1, TCIRG1, TCL2, TCL4, TCL1A, TCN2, TCR1, TCR1, TCR, TCR, TCR1, TCR, TCR TECK, TECTA, TEK, TEL / AML1, TELAB1, TEX15, TF, TFAP2B, TFE3, TFR2, TG, TGFA, TGF-β, TGFBI, TGFB1, TGFBR2, TGFBRE, TGFβ, TGFβRII, TGIF, TGM-4 TH, THAS, THBD, THC, THC2, THM, THPO, THRA, THRB, TIMM8A, TIMP2, TIMP3, TIMP1, TITF1, TKCR TKT, TLP, TLR1, TLR10, TLR2, TLR3, TLR4, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR1, TM4SF1, TM4SF2, TMC1, TMD, TRS, TNF, TRS TNFSF6, TNFα, TNFβ, TNNI3, TNNT2, TOC, TOP2A, TOP1, TP53, TP63, TPA, TPBG, TPI, TPI / m, TPI1, TPM3, TPM1, TPMT, TPO, TPS, TPTA, TRA, TRAG3, TRAPPC2 TRC8, TREH, TRG, TRH, TRIM32, TRIM37, TRP1, TRP2, TRP-2 / 6b, TRP-2 / INT2, Trp-p8, RPS1, TS, TSC2, TSC3, TSC1, TSG101, TSHB, TSHR, TSP-180, TST, TTGA2B, TTN, TTPA, TTR, TU M2-PK, TULP1, TWIST, TYH, TYR, TYROBP, TYROBP, TYRP1 , UBE2A, UBE3A, UBE1, UCHL1, UFS, UGT1A, ULR, UMPK, UMPS, UOX, UPA, UQCRC1, URO5, UROD, UPK1B, UROS, USH2A, USH3A, USH1C, VY, USY1C , VDR, VEGF, VEGFR-2, VEGFR-1, VEGFR-2 / FLK-1, VHL, VIM, VMD2, VMD1, VMGLOM, VNEZ, VNF, VP, V NI, VWF, VWS, WAS, WBS2, WFS2, WFS1, WHCR, WHN, WISP3, WMS, WRN, WS2A, WS2B, WSN, WSS, WT2, WT3, WT1, WTS, WWS, XAGE, XDH, XIC, XST XK, XM, XPA, XPC, XRCC9, XS, ZAP70, ZFHX1B, ZFX, ZFY, ZIC2, ZIC3, ZNF145, ZNF261, ZNF35, ZNF41, ZNF6, ZNF198, ZWS1, or a fragment or variant thereof. Such fragments and variants preferably exhibit sequence homology or sequence identity as defined above.

  Component (B) may also be selected from protein kinase inhibitors, particularly inhibitors of c-Jun amino terminal kinase, which is a protein kinase, ie, JNK inhibitors. In general, as component (B) of the transporter-loading conjugate molecule, a suitable JNK inhibitor may be derived from the human or rat IB1 sequence, preferably SEQ ID NO: 117 (rat IB1 cDNA). Sequence and its predicted amino acid sequence), SEQ ID NO: 118 (describes the rat IB1 protein sequence encoded by the exon-intron boundary of the rIB1 gene-splice donor), SEQ ID NO: 119 (describes the human IB1 protein sequence), Alternatively, it may be derived from the amino acid sequence defined or encoded by any sequence according to SEQ ID NO: 120 (describes the human IB1 cDNA sequence), or any fragment or variant thereof. See above for definitions of fragments and variants.

  A JNK inhibitor sequence suitable as component (B) preferably has a total length of less than 150 amino acid residues, more preferably 5 to 150 amino acid residues, still more preferably 10 to 100 amino acid residues, Particularly preferably 10 to 75 amino acid residues, most preferably 10 to 50 amino acid residues, for example 10 to 30, 10 to 20, or 10 to 15 amino acid residues. It is preferable to contain. Such JNK inhibitor sequences and the above ranges are more preferably any of the JNK inhibitor sequences referred to herein, and even more preferably defined by SEQ ID NO: 119 or encoded by SEQ ID NO: 120. Amino acid sequence, even more preferably, a region between nucleotides 420-980 of SEQ ID NO: 120 or amino acids 105-291 of SEQ ID NO: 119, most preferably nucleotides 561-647 of SEQ ID NO: 120 or amino acids 152-2 of SEQ ID NO: 119 You may select from between 180 regions.

  According to certain embodiments, JNK inhibitor sequences suitable as component (B) generally do at least one of the following: bind to JNK, and at least one JNK-activated transcription factor, such as Inhibits activation of c-Jun or ATF2 (see, eg, SEQ ID NOs: 127 and 128, respectively) or Elk1.

  Similarly, a suitable JNK inhibitor sequence as component (B) comprises at least one amino acid sequence according to any one of SEQ ID NOs: 117 to 200, or a fragment, derivative or variant thereof, or these Preferably it consists of. More preferably, the JNK inhibitor sequence, as used herein, is an amino acid sequence according to SEQ ID NO: 117-200, or a variant, fragment or derivative thereof, 1 copy, 2 copies, 3 copies, 4 copies. Or more. When more than one copy of the amino acid sequence according to SEQ ID NOs: 117 to 200, or a variant, fragment or derivative thereof is present, they may be directly linked to each other without any linker sequence, 1 to 10, Preferably, they may be linked via a linker sequence containing 1 to 5 amino acids. The amino acid forming the linker sequence is preferably selected from glycine or proline as the amino acid residue. More preferably, the amino acid sequence according to SEQ ID NOs: 117-200, or a fragment, derivative or variant thereof, as used herein, is due to a hinge of 2, 3 or more proline residues. They may be separated from each other.

  A suitable JNK inhibitor sequence as component (B) may be composed of L-amino acids, D-amino acids, or a combination of L-amino acids and D-amino acids. Preferably, said JNK inhibitor sequence, as used herein, is at least 1, or even 2, preferably at least 3, 4, or 5, more preferably at least 6, 7 , 8, or 9 and even more preferably at least 10 or more D-amino acids and / or L-amino acids, wherein D-amino acids and / or L-amino acids are used herein as You may arrange | position in the block form, the non-block form, or alternately in the JNK inhibitor arrangement | sequence.

  According to one preferred embodiment, the JNK inhibitor sequence suitable as component (B) may consist entirely of L-amino acids. As used herein, a JNK inhibitor sequence may comprise or consist of at least one “native JNK inhibitor sequence” according to SEQ ID NO: 121 or 123. In this context, “native JNK inhibitor sequence” refers to the unmodified JNK inhibitor sequence according to either SEQ ID NO: 121 or 123, which is composed entirely of D-amino acids.

Thus, component (B) Suitable JNK inhibitor sequence comprises at least one (native) amino acid sequence _{^{NH 2 -X n b -X n a}} -RPTTLXLXXXXXXXQD-X n b -COOH (L-IB generic (s) ) (SEQ ID NO: 123) and / or JIB binding domain (JBD) XRPTTLXLXXXXXXQDS / TX (L-IB (generic)) (SEQ ID NO: 131) of IB1, or may be composed of the above sequence. In this situation, each X generally represents an amino acid residue and is preferably selected from any (native) amino acid residue. X _n ^a generally represents one amino acid residue, and is preferably selected from any amino acid residue except serine or threonine, and n (the number of X repeats) is 0 or 1. Additionally, each _X ^{n b} may be selected from any amino acid residue, n (number of iterations X) is 0～5,5～10,10～15,15～20,20～30, or That's it. However, when n of X _n ^a (number of iterations X) is 0, X _n ^b, to avoid the presence of a serine or threonine at the position of X _n ^a, include serine or threonine at the C-terminus Preferably not. Preferably, X _n ^b represents a continuous (poly) peptide residue derived from SEQ ID NO: 121 or 123. X _n ^a and X _n ^b may represent either a D amino acid or an L amino acid. Further, the JNK inhibitor sequence as used herein comprises at least one (native) amino acid sequence selected from the group comprising the JNK binding domain DTYRPKRPTTNLNLFPQVPRSQDT (L-IB1) (SEQ ID NO: 129) of IB1. May be included, or may be composed of the arrangement. More preferably, the JNK inhibitor sequence as used herein further comprises at least one (native) amino acid sequence NH ₂ -RPKRPTTLNLFPQVPRSQD-COOH (L-IB1 (s)) (SEQ ID NO: 121). Or it may consist of the arrangement. Further, as used herein, the JNK inhibitor sequence may comprise or consist of at least one (native) amino acid sequence selected from the group comprising the following IB1 JNK binding domain: may _{be: L-IB1 (s1) (} NH 2 -TLNLFPQVPRSQD-COOH, SEQ ID NO: _{133); L-IB1 (s2} ) (NH 2 -TTLNLFPQVPRSQ-COOH, SEQ ID NO: 134); L-IB1 (s3 ) ( NH ₂ -PTTLNLFPQVPRS-COOH, SEQ ID NO: _{135); L-IB1 (s4} ) (NH 2 -RPTTLNLFPQVPR-COOH, SEQ ID NO: _{136); L-IB1 (s5} ) (NH 2 -KRPTTLNLFPQVP-COOH, SEQ ID NO: 137); _{L-IB1 (s6) (NH} 2 -PKRP TLNLFPQV-COOH, SEQ ID NO: _{138); L-IB1 (s7} ) (NH 2 -RPKRPTTLNLFPQ-COOH, SEQ ID NO: _{139); L-IB1 (s8} ) (NH 2 -LNLFPQVPRSQD-COOH, SEQ ID NO: 140); L-IB1 _{(s9) (NH 2 -TLNLFPQVPRSQ-} COOH, SEQ ID NO: _{141); L-IB1 (s10} ) (NH 2 -TTLNLFPQVPRS-COOH, SEQ ID NO: _{142); L-IB1 (s11} ) (NH 2 -PTTLNLFPQVPR-COOH, SEQ No. _{143); L-IB1 (s12} ) (NH 2 -RPTTLNLFPQVP-COOH, SEQ ID NO: _{144); L-IB1 (s13} ) (NH 2 -KRPTTLNLFPQV-COOH, SEQ ID NO: 145); L-IB1 (s14 ) (NH ₂ -PKRPTTLNLFPQ-COOH, SEQ ID NO: _{146); L-IB1 (s15} ) (NH 2 -RPKRPTTLNLFP-COOH, SEQ ID NO: _{147); L-IB1 (s16} ) (NH 2 -NLFPQVPRSQD-COOH, SEQ ID NO: 148); L _{-IB1 (s17) (NH 2 -LNLFPQVPRSQ} -COOH, SEQ ID NO: _{149); L-IB1 (s18} ) (NH 2 -TLNLFPQVPRS-COOH, SEQ ID NO: _{150); L-IB1 (s19} ) (NH 2 -TTLNLFPQVPR-COOH , SEQ ID NO: _{151); L-IB1 (s20} ) (NH 2 -PTTLNLFPQVP-COOH, SEQ ID NO: _{152); L-IB1 (s21} ) (NH 2 -RPTTLNLFPQV-COOH, SEQ ID NO: 153); L-IB1 (s22 ) ( NH ₂ -KRPTTLNLFPQ-COOH, SEQ ID NO: _{154); L-IB1 (s23} ) (NH 2 -PKRPTTLNLFP-COOH, SEQ ID NO: _{155); L-IB1 (s24} ) (NH 2 -RPKRPTTLNLF-COOH, SEQ ID NO: 156); L-IB1 (s25) (NH ₂ -LFPQVPRSQD-COOH, SEQ ID NO: 157); L-IB1 (s26) (NH ₂ -NLFPQVPRSQ-COOH, SEQ ID NO: 158); L-IB1 (s27) (NH ₂ -LNLFPQVPRS- COOH, SEQ ID NO: _{159); L-IB1 (s28} ) (NH 2 -TLNLFPQVPR-COOH, SEQ ID NO: _{160); L-IB1 (s29} ) (NH 2 -TTLNLFPQVP-COOH, SEQ ID NO: 161); L-IB1 (s30 ) (NH ₂ - TTLNLFPQV-COOH, SEQ ID NO: _{162); L-IB1 (s31} ) (NH 2 -RPTTLNLFPQ-COOH, SEQ ID NO: _{163); L-IB1 (s32} ) (NH 2 -KRPTTLNLFP-COOH, SEQ ID NO: 164); L-IB1 _{(s33) (NH 2 -PKRPTTLNLF-} COOH, SEQ ID NO: 165); and _{L-IB1 (s34) (NH} 2 -RPKRPTTLNL-COOH, SEQ ID NO: 166).

  Furthermore, suitable JNK inhibitor sequence component (B), the IB1 (long) JNK binding domain PGTGCGDTYRPKRPTTLNLFPQVPRSQDT (IB1-long) (SEQ ID NO: 125), of IB2 (long) JNK binding domain IPSPSVEEPHKHRPTTLRLTTLGAQDS (IB2-long) ( SEQ ID NO: 126), c-Jun's JNK binding domain GAYGYSNPKILKQSMTLNLADPVGNLKPH (c-Jun) (SEQ ID NO: 127), ATF2 JNK binding domain TNEDHLAVHKHKHEMTLKFGPARNDSVIV (ATF2) (SEQ ID NO: 128) selected from the group comprising at least one (SEQ ID NO: 128) A) may comprise an amino acid sequence or may consist of said sequence. In this situation, the alignment reveals the 8 amino acid sequences that are partially conserved, and by further comparing the JBD of IB1 and IB2, the 7 that are highly conserved between the two sequences and Two blocks of three amino acids were revealed.

  A suitable JNK inhibitor sequence as component (B) may be partially or fully composed of D-amino acids. These JNK inhibitor sequences consisting of D-amino acids are preferably non-native D-retro-inverso sequences of the above (native) JNK inhibitor sequences.

  The term “retroinverso sequence” refers to an isomer of a linear (poly) peptide sequence in which the direction of the sequence is reversed and the chirality of each amino acid residue is also reversed (see, eg, Jameson et al. , Nature, 368, 744-746 (1994); Brady et al., Nature, 368, 692-693 (1994)). The advantage of combining the D-enantiomer and reverse synthesis is that the position of the carbonyl group and amino group in each amide bond is interchanged, but the position of the side chain group at each α carbon is preserved. Unless otherwise specified, any given L-amino acid sequence or (poly) peptide, when used in accordance with the present invention, is the reverse sequence or (poly) of the corresponding native L-amino acid sequence or (poly) peptide. Assume that the peptide can be synthesized and converted to a D-retroinverso sequence or (poly) peptide. In contrast, the term “reverse sequence” refers to a sequence in which the orientation of the sequence is reversed, but the chirality of each amino acid residue is not reversed (eg, D-Arg-L-Arg-L-Arg → L-Arg-L-Arg-D-Arg).

Therefore, transporters present invention - Suitable JNK inhibitor sequence as component (B) of the cargo conjugate molecule, the amino acid sequence _{^{NH 2 -X n b -DQXXXXXXXLXLTTPR-X}} n a -X n b -COOH (D- IB1 generic (s)) (SEQ ID NO: 124) and / or XS / TDQXXXXXXXLXLTPRX (D-IB (generic)) (SEQ ID NO: 132) or may comprise at least one D-retroinverso sequence May be configured. When used in this context, X, X _n ^a , and X _n ^b are as defined above (preferably representing D amino acids), and X _n ^b is preferably contiguous SEQ ID NO: 122 Or a residue from 124. Further, a JNK inhibitor sequence, as used herein, is at least one D-retroinverso form according to an amino acid sequence comprising the JNK binding domain (JBD) TDQSRPVQPFLNLLTTPRKPRYTD (D-IB1) (SEQ ID NO: 130) of IB1. An array may be included, or may consist of the array. More preferably, said JNK inhibitor sequence, as used herein, the amino acid sequence _{NH 2 -DQSRPVQPFLNLTTPRKPR-COOH (D-} IB1 (s)) ( SEQ ID NO: 122) in accordance at least one D- retro-inverso It may contain a type array or may consist of said array. Furthermore, the JNK inhibitor sequence, as used herein, may comprise at least one D-retroinverso sequence according to the amino acid sequence comprising the following IB1 JNK binding domain (JBD), May be configured: D-IB1 (s1) (NH ₂ -QPFLNLTTPRKPR-COOH, SEQ ID NO: 167); D-IB1 (s2) (NH ₂ -VQPFLNLTTPRKP-COOH, SEQ ID NO: 168); D-IB1 (s3) _(NH 2 -PVQPFLNLTTPRK-COOH, SEQ ID NO: _{169); D-IB1 (s4} ) (NH 2 -RPVQPFLNLTTPR-COOH, SEQ ID NO: _{170); D-IB1 (s5} ) (NH 2 -SRPVQPFLNLTTP-COOH, SEQ ID NO: 171) _{; D-IB1 (s6) (} NH 2 -QSR VQPFLNLTT-COOH, SEQ ID NO: _{172); D-IB1 (s7} ) (NH 2 -DQSRPVQPFLNLT-COOH, SEQ ID NO: _{173); D-IB1 (s8} ) (NH 2 -PFLNLTTPRKPR-COOH, SEQ ID NO: 174); D-IB1 _{(s9) (NH 2 -QPFLNLTTPRKP-} COOH, SEQ ID NO: _{175); D-IB1 (s10} ) (NH 2 -VQPFLNLTTPRK-COOH, SEQ ID NO: _{176); D-IB1 (s11} ) (NH 2 -PVQPFLNLTTPR-COOH, SEQ D-IB1 (s12) (NH ₂ -RPVQPFLNLTTP-COOH, SEQ ID NO: 178); D-IB1 (s13) (NH ₂ -SRPVQPFLNLTTT-COOH, SEQ ID NO: 179); D-IB1 (s14) (N H ₂ -QSRPVQPFLNLT-COOH, SEQ ID NO: _{180); D-IB1 (s15} ) (NH 2 -DQSRPVQPFLNL-COOH, SEQ ID NO: _{181); D-IB1 (s16} ) (NH 2 -FLNLTTPRKPR-COOH, SEQ ID NO: 182); _{D-IB1 (s17) (NH} 2 -PFLNLTTPRKP-COOH, SEQ ID NO: _{183); D-IB1 (s18} ) (NH 2 -QPFLNLTTPRK-COOH, SEQ ID NO: _{184); D-IB1 (s19} ) (NH 2 -VQPFLNLTTPR- COOH, SEQ ID NO: _{185); D-IB1 (s20} ) (NH 2 -PVQPFLNLTTP-COOH, SEQ ID NO: _{186); D-IB1 (s21} ) (NH 2 -RPVQPFLNLTT-COOH, SEQ ID NO: 187); D-IB1 (s22 ) _(NH 2 -SRPVQPFLNLT-COOH, SEQ ID NO: _{188); D-IB1 (s23} ) (NH 2 -QSRPVQPFLNL-COOH, SEQ ID NO: _{189); D-IB1 (s24} ) (NH 2 -DQSRPVQPFLN-COOH, SEQ ID NO: 190) _{; D-IB1 (s25) (} NH 2 -DQSRPVQPFL-COOH, SEQ ID NO: _{191); D-IB1 (s26} ) (NH 2 -QSRPVQPFLN-COOH, SEQ ID NO: _{192); D-IB1 (s27} ) (NH 2 -SRPVQPFLNL -COOH, SEQ ID NO: _{193); D-IB1 (s28} ) (NH 2 -RPVQPFLNLT-COOH, SEQ ID NO: _{194); D-IB1 (s29} ) (NH 2 -PVQPFLNLTT-COOH, SEQ ID NO: 195); D-IB1 ( s30) (NH ₂ VQPFLNLTTP-COOH, SEQ ID NO: _{196); D-IB1 (s31} ) (NH 2 -QPFLNLTTPR-COOH, SEQ ID NO: _{197); D-IB1 (s32} ) (NH 2 -PFLNLTTPRK-COOH, SEQ ID NO: 198); D-IB1 _{(s33) (NH 2 -FLNLTTPRKP-} COOH, SEQ ID NO: 199); and _{D-IB1 (s34) (NH} 2 -LNLTTPRKPR-COOH, SEQ ID NO: 200).

  Exemplary JNK inhibitor sequences suitable as component (B) are shown in Tables 5-8 (SEQ ID NOs: 121-200). This table shows the name of the JNK inhibitor sequence as used herein, the sequence identification number, length, and amino acid sequence. Further, Tables 5 to 8 show IB1-derived sequences and general formulas thereof, such as SEQ ID NOS: 121 and 122 and SEQ ID NOS: 123 and 124, respectively. Tables 5-8 further disclose L-IB1 sequences according to SEQ ID NOs 133-166 and D-IB1 sequences according to SEQ ID NOs 167-200.

  A JNK inhibitor sequence suitable as component (B) further comprises at least one variant, fragment, and / or native amino acid sequence as defined above or a non-native amino acid sequence according to SEQ ID NO: 121-200. Derivatives may be included, or may be composed of these. These variants, fragments, and / or derivatives, as used herein, are native or non-native JNK inhibitor sequences disclosed above, particularly the native or non-native amino acids according to SEQ ID NOs: 121-200. It is preferred to retain the biological function / activity of the sequence, ie to do at least one of the following: bind to JNK and at least one JNK-activating transcription factor, eg c-Jun, ATF2, Or inhibits activation of Elk1 (see above for functional / activity tests).

  Effector molecules suitable as component (B) are furthermore antigens or antigen fragments, preferably protein and (poly) peptide antigens such as tumor antigens or antigen fragments thereof, allergen antigens or antigen fragments thereof, autoimmune self antigens or An antigen fragment thereof, a pathogenic antigen or an antigen fragment thereof, derived from a virus, preferably an antigen derived from the following virus or an antigen fragment thereof: cytomegalovirus (CMV), orthopox variola virus, orthopox aristrim virus, sheep Parapox virus, infectious molluscumoma virus, herpes simplex virus type 1, herpes simplex virus type 2, herpes B virus, varicella zoster virus, pseudorabies virus, human giant cell virus, human herpes virus type 6, human herpes virus Type 7, Epstein-Berui , Human herpesvirus type 8, hepatitis B virus, chikungunya virus, onyonyon virus, ruby virus, hepatitis C virus, GB virus C, West Nile virus, dengue virus, yellow fever virus, jumping disease virus, St. Louis encephalitis virus , Japanese encephalitis virus B, Powassan virus, FSME virus, SARS, SARS-related coronavirus, human coronavirus 229E, human coronavirus Oc43, torovirus, human T lymphocyte tropic virus type I, human T lymphocyte tropism Virus type II, HIV (AIDS), ie human immunodeficiency virus type 1 or human immunodeficiency virus type 2, influenza virus, Lassa virus, lymphocytic choriomeningitis virus, Tacaribe virus, Junin virus, Machupoui , Borna disease virus, Bunyumwela virus, California encephalitis virus, Rift Valley fever virus, Pterfly fever virus, Tuscany virus, Crimea-Congo hemorrhagic fever virus, Hazara virus, Hasan virus, Hantan virus, Seoul virus, Prospect Hill virus, Pumara virus, Dobrava beograd virus, Tula virus, Shin nonbull virus, Lake Victoria Marburg virus, Zaire Ebola virus, Sudan Ebola virus, Cote d'Ivoire Ebola virus, Influenza virus type A, Influenza virus type B, Influenza virus C, parainfluenza virus, malaria virus, Marburg virus, measles virus, epidemic parotitis virus, RS virus Irs, human metapneumovirus, vesicular stomatitis Indiana virus, rabies virus, mocola virus, dubenheage virus, European bat lyssa virus type 1 + 2, Australian bat lyssa virus, adenovirus type A-F, human papilloma virus, condyloma virus type 6, condyloma Selected from virus type 11, polyoma virus, adeno-associated virus type 2, rotavirus, orbivirus, chickenpox including varicella-zoster, or antigens derived from leishmania, trypanosoma, amoeba, bacteria, or antigen fragments thereof Alternatively, it may be selected from epitopes or variants of the antigen or antigen fragment thereof. Fragments and variants of the antigens defined above are about 10%, about 20%, about 30%, about 40%, about 50% with one of the antigens or antigen fragments shown above or described above Preferably about 60%, about 70%, about 80%, or about 90% sequence homology or sequence identity. In this situation, the fragment and variant definitions already defined above apply as well. Further encompassed are epitopes (also referred to as “antigenic determinants”) of the antigen or antigen fragment defined above.

Furthermore, effector molecules suitable as component (B) of the transporter-loading conjugate molecule may be selected from antibodies. According to the present invention, such an antibody is any antibody known in the art, such as any recombinantly produced or natural antibody, particularly suitable for therapeutic, diagnostic or scientific purposes. Or an antibody identified in connection with a particular cancer disease. As used herein, the term “antibody” is used in the broadest sense and specifically includes monoclonal and polyclonal antibodies (including agonists, antagonists, and blocking or neutralizing antibodies), as well as multi-epitope specificity. Including the antibody species. In accordance with the present invention, an “antibody” is generally any antibody known in the art (eg, IgM, IgD, IgG, IgA, and IgE antibodies), eg, a natural antibody, immunizing a host organism. By recombination using antibodies produced by immunization, antibodies isolated and identified from antibodies produced by immunizing natural antibodies or host organisms, and molecular biology methods known in the art In addition to the antibodies produced, etc., chimeric antibodies, human antibodies, humanized antibodies, bispecific antibodies, intrabodies, ie antibodies that are expressed in cells and optionally are localized in specific intracellular compartments, As well as fragments and variants of the aforementioned antibodies. In general, an antibody consists of a light chain and a heavy chain, both of which have a variable domain and a constant domain. The light chain consists of an N-terminal variable domain V _L and a C-terminal constant domain C _L. In contrast, the heavy chain of an IgG antibody consists of, for example, an N-terminal variable domain V _H and three constant domains C _H 1, C _H 2, and C _H 3. The antibodies, in this situation, including fragments and variants of the antibody, e.g., F _ab fragments, and F _c fragment. Such fragments and variants may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% with one of the above antibodies. Preferably exhibit sequence homology or sequence identity. In this situation, the fragment and variant definitions defined above apply as well.

Furthermore, component (B) of the transporter-loading conjugate molecule of the present invention comprises an apoptosis factor or apoptosis-related protein comprising: AIF, Apaf, such as Apaf-1, Apaf-2, Apaf-3, order APO- 2 (L), APO-3 (L), apopain, Bad, Bak, Bax, Bcl-2, Bcl-x _L , Bcl-x _S , bik, Bok, CAD, calpain, caspase, for example, caspase-1, Caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, ced-3, ced-9, c- Jun, c-Myc, crmA, cytochrome C, CdR1, DcR1, DD, DED, DISC, _{NA-PK CS, DR3, DR4} , DR5, FADD / MORT-1, FAK, Fas (Fas- ligand CD95 / fas (receptor)), FLICE / MACH, FLIP , fodrin, fos, G-actin, Gas-2 , gelsolin, granzyme A / B, ICAD, ICE, JNK, lamin A / B, MAP, Max, MCL-1, Mdm-2, MEKK-1, MORT-1, Myd88, NEDD, NF- κ B, NuMa, p38, p53, PAK-2, PARP, perforin, PITSLRE, PKC delta, pRb, presenilin, prICE, RAIDD, Ras, RIP, sphingomyelinase, herpes simplex thymidine kinase, TRADD, TRAF2, TRAIL-R1, TRAIL-R2 , TRAIL-R3 , Transglutaminase or the like, or a fragment or variant thereof, or a component of the wnt-signaling pathway such as β-catenin, ICF-family, polo-like kinase, CiP2A, PP2A, or a fragment or variant thereof May be. Such fragments and variants may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% with one of the sequences shown or described above. %, About 80%, or about 90% sequence homology or sequence identity is preferred. In this situation, the fragment and variant definitions defined above apply as well.

  Effector molecules suitable as component (B) of the transporter-loading conjugate molecule are further selected from at least one or more partial length or full length BH3 domains and / or at least one partial length or full length BH3-only protein. May be. In this context, the BH3-only protein is preferably defined as a member of the Bcl-2 family that represents a regulator of apoptosis by interacting with other members of the Bcl-2 family. In the context of the present invention, component (B) of the transporter-loading conjugate molecule comprises at least one or more partial length or full length BH3 domain sequences of BH3-only protein, or partial length or full length BH3-only protein (Bcl). -Amino acid sequences, defined as subclasses of -2 family proteins), which interact with at least one Bcl-2 family protein or at least one apoptosis of the Bcl-2 family By activating or sensitizing the facilitating member, apoptosis can be induced. These functional activities can be tested by assaying for pro-apoptotic activity by a suitable assay method, eg, a binding assay or by an apoptosis assay. Preferably, the amino acid sequence used as component (B) of the transporter-loading conjugate molecule of the invention has at least one partial or full length BH3 domain sequence and / or Bid, Bad, Noxa, Puma, Bim, It may comprise or consist of at least one partial-length or full-length BH3-only protein sequence selected from the group consisting of Bik, Bmf, DP5 / Hrk and Bok. Alternatively, the amino acid sequence used as component (B) of the transporter-loading conjugate molecule of the present invention comprises at least one partial length or full length BH3 domain sequence, and / or at least one partial length or full length BH3-only. It may comprise a combination of protein sequences or may consist of said combination, said combination preferably selected from the group consisting of, for example: Bid and Bad, Bim and Bad, Bik and Bad, Puma And Bad, Noxa and Bad, Bmf and Bad, DP5 / Hrk and Bad, Bok and Bad, Bik and Bim, Bik and Bid, Bik and Puma, Bik and Noxa, Bik and Bmf, Bik and DP5 / Hrk, Bik and Bok , Bid and Puma, Bid and Noxa, Bid Bim, Bid and Bmf, Bid and DP5 / Hrk, Bid and Bok, Bim and Noxa, Bim and Puma, Bim and Bmf, Bim and DP5 / Hrk, Bim and Bok, Puma and Noxa, Puma and Bmf, Puma and DP5 / Hrk, Puma and Bok, Noxa and Bmf, Noxa and DP5 / Hrk, Noxa and Bok. The BH3 sequence or BH3-only protein sequence defined above (full length or partial length) may be selected, for example, from any mammalian BH3-only protein, in particular a human isoform. Accordingly, component (B) of the transporter-loading conjugate molecule of the invention has at least one partial or full length BH3 domain as defined by any of SEQ ID NOs: 201-217 (see Tables 9-10). The sequence and / or at least one BH3-only protein sequence may be included, or may consist of said sequence. Preferably, the amino acid sequence used as component (B) of the transporter-loading conjugate molecule of the present invention is further at least one partial length or full length BH3 domain defined by any of SEQ ID NOs: 201-217 It may comprise or consist of at least one fragment or variant of a sequence and / or at least one BH3-only protein sequence. Such fragments and variants have a sequence length of less than 50 amino acids, preferably less than 40 amino acids, more preferably less than 30 amino acids, or one of the sequences set forth above or any one of the sequences set forth in SEQ ID NOs: 201-217 Preferably, it exhibits about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% sequence homology or sequence identity. In this situation, the fragment and variant definitions defined above apply again. Furthermore, native sequence fragments or variants generally comprise a BH3 domain sequence or at least partially comprise a BH3 domain sequence (at least 7 amino acids of the BH3 domain sequence).

  Protein or (poly) peptide sequences described herein that can be used as component (B), eg, therapeutically active proteins, antigens, antibodies, apoptotic factors, proteases involved in disease states, preferably peptides Sequences such as protease inhibitors, BH3 domains, etc. are provided as protein or (poly) peptide sequences that are either native forms consisting of L-amino acids or are retro-inverso D forms consisting of (all) D-amino acids. This means that these sequences must be inverted by reversing the ends, i.e. the native C-terminus becomes the inverted N-terminus and the native N-terminus is inverted. Becomes the C-terminal. Alternatively, these protein or (poly) peptide sequences described above may provide the protein or (poly) peptide sequence as a mixture of L-amino acids and D-amino acids.

  Component (B) is also a nucleic acid, preferably a protein or (poly) peptide as defined above, eg, a therapeutically active protein and (poly) peptide, an antigen, an antibody, an apoptotic factor, a protease involved in a disease state Preferably, it may be selected from a nucleic acid encoding a peptide protease inhibitor, a BH3 domain, or a partial or full-length BH3-only protein, or a variant of these fragments. In this situation, the nucleic acid preferably comprises a single stranded, double stranded or partially double stranded nucleic acid, preferably genomic DNA, cDNA, RNA, siRNA, antisense DNA, antisense RNA, microRNA. , Ribozymes, complementary RNA / DNA sequences with or without expression elements, minigenes, gene fragments, regulatory elements, promoters, and combinations thereof.

  As a further specific example, component (B) may be selected from siRNA. In this situation, siRNAs specifically related to the phenomenon of RNA interference are targeted. In the course of immunological research, the phenomenon of RNA interference was found. In recent years, RNA-based defense mechanisms have been discovered, which occur both in the fungal kingdom, in the plant kingdom and in the animal kingdom, and act as a “genomic immune system”. The genomic immune system was originally reported in various species independently of each other (initially C. elegans) and later confirmed that the mechanisms underlying these processes were identical: RNA-mediated virus resistance, plant PTGS (post-transcriptional gene silencing), and eukaryotic RNA interference are based on common mechanisms. In vitro technology of RNA interference (RNAi) is based on double-stranded RNA molecules (dsRNA) that induce sequence-specific suppression of gene expression (Zamore (2001) Nat. Struct. Biol. 9: 746-750; Sharp). (2001) Genes Dev. 5: 485-490; Hannon (2002) Nature 41: 244-251). When long dsRNAs are transfected into mammalian cells, protein kinase R and RnaseL are activated, causing non-specific effects such as, for example, interferon response (Stark et al. (1998) Annu. Rev. Biochem. 67. : 227-264; He and Katze (2002) Viral Immunol. 15: 95-119). Since siRNA shorter than 30 bp does not induce the non-specific action, for example, when using a short RNA of 21 mer to 23 mer, so-called siRNA (small interfering RNA), these non-specific actions are avoided (Elbashir et al. (2001) Nature 411: 494-498). Recently, dsRNA molecules have also been used in vivo (McCaffrey et al. (2002), Nature 418: 38-39; Xia et al. (2002), Nature Biotech. 20: 1006-1010; Brummelkamp et al. (2002). ), Cancer Cell 2: 243-247). Therefore, siRNA used as an effector molecule suitable as component (B) of the transporter-loading conjugate molecule of the present invention generally has about 8 nucleotides to about 30 nucleotides, preferably about 17 nucleotides to about 25 nucleotides. More preferably 20 to 25 nucleotides, particularly preferably 21 to 23 nucleotides (single-stranded or) double-stranded RNA sequences, preferably double-stranded RNA sequences. In principle, all sections having a length of 17 base pairs to 29 base pairs, preferably 19 base pairs to 25 base pairs, more preferably 21 base pairs to 23 base pairs occurring in the coding region of the above-mentioned protein (sequence) , Can function as a target sequence of siRNA. Similarly, siRNA can also target the nucleotide sequence of the aforementioned protein (sequence) that is not present in the coding region, in particular the 5 'non-coding region of RNA, for example, the non-coding region of RNA having a regulatory function. Accordingly, the target sequence of siRNA may be present in at least one of the translated and / or untranslated region of RNA and the region of regulatory element. Moreover, the target sequence of siRNA may be present in the overlapping region of the untranslated region and the translated region, and specifically, the target sequence may include at least one nucleotide upstream of the start codon of the coding region. .

  As another specific example, component (B) may be selected from antisense RNA. In this situation, antisense RNA is a (single-stranded) RNA molecule that is transcribed based on the coding strand of (genomic) DNA, rather than a template, so as to be complementary to sense (messenger) RNA. It is preferable. Antisense RNAs suitable as component (B) of the transporter-loading conjugate molecules of the present invention generally form a duplex between the sense RNA molecule and the antisense RNA molecule, thereby responding Translation of mRNA can be blocked. As used herein, an antisense RNA is any portion of an mRNA sequence derived from, for example, genomic DNA, where translation of the encoded protein or (poly) peptide is reduced / suppressed by the antisense RNA. And protein (poly) peptides defined herein (eg, the therapeutically active proteins and (poly) peptides, antigens, antibodies, apoptotic factors, proteases involved in the pathology, preferably peptide protease inhibitors, BH3 -Targeting at least one of any part of an mRNA sequence capable of encoding any protein such as a domain, a partial length BH3-only protein, a full-length BH3-only protein, or a variant of these fragments) It may be. Therefore, the target sequence of the antisense RNA in the target mRNA (or target (genomic) DNA) is the translated and / or untranslated region of the mRNA (or target (genomic) DNA), for example, the region of the regulatory element In particular, it may be present in the 5′-non-coding region of mRNA (or target (genomic) DNA) that exerts a regulatory function. In addition, the target sequence of the antisense RNA in the target mRNA (or target (genomic) DNA) is the untranslated sequence and translation (coding) sequence of the target mRNA (or target (genomic) DNA). By including a region that is partially complementary to the antisense RNA can be constructed to bind to mRNA (or target (genomic) DNA); specifically, the antisense RNA is The target mRNA (or target (genomic) DNA) may be complementary up to at least one nucleotide upstream of the start codon of the coding region of the target mRNA. Preferably, as used herein, antisense RNA is from about 5 nucleotides to about 5,000 nucleotides, preferably from about 500 nucleotides to about 5,000 nucleotides, more preferably from about 1,000 nucleotides to about 5, 000 nucleotides, or about 5 nucleotides to about 1,000 nucleotides, about 5 nucleotides to about 500 nucleotides, about 5 nucleotides to about 250 nucleotides, about 5 nucleotides to about 100 nucleotides, about 5 nucleotides to about 50 nucleotides, or about 5 nucleotides From about 20 nucleotides to about 100 nucleotides, from about 20 nucleotides to about 80 nucleotides, or from about 20 nucleotides to about 60 nucleotides.

  As a further specific example, the component (B) may be a pharmaceutical selected from, for example, a cytotoxic drug or an antitumor drug suitable as a chemotherapeutic drug. In general, chemotherapeutic drugs suitable as component (B) can be divided into three main classes based on the mechanism of action. The chemotherapeutic agents can (a) stop the synthesis of the basic units of pre-DNA molecules: these agents function in a number of different ways. The basic units of DNA are folic acid, heterocyclic bases, and nucleotides, which are naturally produced intracellularly. All of these agents have the function of blocking several steps of nucleotide or deoxyribonucleotide formation (essential for DNA production). If these steps are blocked, nucleotides that are basic units of DNA and RNA cannot be synthesized. Therefore, cells cannot replicate DNA because it cannot produce DNA without nucleotides. Examples of this class of drugs include methotrexate (Abitrexate®), fluorouracil (Adrucil®), hydroxyurea (Hydrea®), and mercaptopurine (Purinethol®), thioguanine, Tocopherols may be mentioned, or more generally any nucleotide analogs such as 2'-deoxycytidine analogs. Alternatively, chemotherapeutic agents can (b) directly damage the DNA in the nucleus of the cell. These agents cause chemical damage to DNA and RNA. These disrupt DNA replication and stop replication completely, or cause the production of nonsense DNA or RNA (ie, the newly generated DNA or RNA does not encode anything useful) . Examples of this class of drugs include cisplatin (Platinol®), an anthracycline antitumor agent class (this member can be used as component (B) of the transporter-loading conjugate molecule of the present invention. And daunorubicin (Cerubidin (registered trademark)), doxorubicin (Adriamycin (registered trademark)), and etoposide (VePesid (registered trademark)), or any intercalator. Finally, chemotherapeutic agents can affect (c) the synthesis or disruption of the mitotic spindle: the mitotic spindle is the cell's cell when it begins to divide into two new cells. It functions as a molecular track for the “South and North Pole”. These spindles are very important because they aid in the division of the newly copied DNA so that the copy moves to each of two new cells during cell division. These drugs interfere with cell formation because they disrupt spindle formation. Examples of drugs in this mitotic disruptor class include vinblastine (Velban®), vincristine (Oncovin®), and paclitaxel (Taxol®). . Component (B) of the transporter-load conjugate molecule of the present invention can act according to one of the above mechanisms of action. In other words, each class of anti-tumor drugs, ie, alkylating agents, nitrosoureas, antimetabolites, plant alkaloids, anti-tumor antibiotics, and steroid hormones, is a component of the transporter-load conjugate molecule of the present invention ( B) can be used. To explain in more detail the classification of these drugs, it is emphasized that each anti-cancer drug can be classified according to the effects on the cell cycle and cytochemistry disclosed above. Alkylating agents kill cells by directly attacking DNA. Alkylating agents can be used to treat chronic leukemia, Hodgkin's disease, lymphoma, and certain carcinomas of the lung, breast, prostate, and ovary. Cyclophosphamide is an example of a commonly used alkylating agent. Nitrosoureas act similarly to alkylating agents and also inhibit changes essential for DNA repair. Because these agents cross the blood brain barrier, they are used to treat brain tumors, lymphomas, multiple myeloma, and malignant melanoma. Carmustine and lomustine are the main drugs in this class. Antimetabolites are agents that block cell proliferation by interfering with certain activities, usually DNA synthesis. Once taken up by cells, the antimetabolite stops normal development and replication. All of this class of drugs affects cells during the “S” phase of the cell cycle. Antimetabolites can be used to treat acute and chronic leukemia, chorioepithelioma, and several tumors of the gastrointestinal tract, breast, and ovary. Examples of commonly used antimetabolites are 6-mercaptopurine and 5-fluorouracil (5FU). Antitumor antibiotics are a diverse group of compounds. In general, anti-tumor antibiotics act by binding to DNA and blocking RNA synthesis. These agents are widely used in the treatment of various cancers. The most commonly used drugs in this group are doxorubicin (adriamycin), mitomycin-C, and bleomycin. Plant (vinca) alkaloids are antitumor agents derived from plants. These agents specifically act by blocking cell division during mitosis. Plant alkaloids are commonly used in the treatment of acute lymphoblastic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, neuroblastoma, Wilms tumor, and lung, breast, and testicular cancer. Vincristine and vinblastine are commonly used agents in this group. Steroid hormones are useful in the treatment of several types of tumors. This classification includes adrenocorticosteroids, estrogens, antiestrogens, progesterone, and androgens. Although the specific mechanism of action is not clear, steroid hormones regulate the growth of certain hormone-dependent cancers. Tamoxifen is an example, which is used for estrogen-dependent breast cancer. All species of the tumor can be treated with a transporter-loading conjugate molecule of the invention comprising any of the antitumor agents as component (B).

  One group of cytotoxic or anti-tumor agents that can be used as effector molecules for component (B) of the transporter-loading conjugate molecule of the present invention are alkylating agents, antimetabolites, cytostatic agents Or selected from drugs related to hormone therapy. In this situation, it is preferable to select metal compounds, in particular platinum (derivatives) and taxol classification, as cytotoxic or antitumor agents. Specifically, the drug moiety is, for example, cisplatin, transplatin, satraplatin, oxaliplatin, carboplatin, nedaplatin, chlorambucil, cyclophosphamide, melphalan, azathioprine, fluorouracil, (6) -mercaptopurine, methotrexate, nandrolone, Aminoglycoside, medroxyprogesterone, megestrol acetate, procarbazine, docetaxel, paclitaxel, irinotecan, epipodophyllotoxin, podophyllotoxin, vincristine, vinblastine, docetaxel, daunomycin, daunorubicin, doxorubicin, mitozantrone, topotem , Gemcitabine, fludarabine, navelbine and 5-FUDR. The classification of metal-containing anticancer drugs, for example the classification of platinum compounds, is particularly preferred.

  Further cytotoxic or anti-tumor agents (identified by a generic name) that can be used as component (B) of the transporter-load conjugate molecule of the present invention include alitretinoin, altretamine, azathioprine, bicalutamide, busulfan , Capecitabine, cyclophosphamide, exemestane, letrozole, finasteride, megestrol acetate, triptorelin, temozolomide, mifepristone, tretinoin, oral, tamoxifen, teniposide, imatinib (Gleevec®), gefitinib (IRESSA®) Trademark)), pepromycin sulfate, or camptothecin.

  Another group of cytotoxic drugs or antitumor drugs that can be used as component (B) includes indolocarbazole compounds and the like, for example, staurosporine (and analogs thereof) and rebeccamycin and the like. . As the component (B), a compound belonging to the class of aminoquinazoline (for example, gefitinib) is particularly preferable.

  A further group of cytotoxic or antitumor agents that can be used as effector molecules for component (B) of the transporter-loading conjugate molecule of the present invention include topoisomerase inhibitors such as irinotecan, or mitosis. It may be further selected from kinesin or DHFR.

Furthermore, cytotoxic or antitumor agents that can be used as effector molecules for component (B) of the transporter-loading conjugate molecule of the present invention include, for example, cell growth inhibitory or stimulating factor (PDGF), cells CMGC kinase family (CDK (Cyclin Dependent Kinase), MAPK, etc.), including members of the internal pathway, RAS / RAF signaling pathway, RAF / MEK / ERK signaling pathway (eg RAF-1) or mitogen activated protein kinase pathway GSK3, including CLK), PKA, PKG, Ser / Thr kinase belonging to AGC kinase family including PKC kinase family, vascular endothelial growth factor receptor (VEGFR) -2, VEGFR-3, platelet-derived growth factor receptor β, etc. In addition, new blood vessels Receptor tyrosine kinases involved in tumor progression, Flt-3, (including ET-1, ET-2, ET-3) Endothelin (ET) system, ET _A receptor _(ET A R) and _ET B R For example, c-KIT targeted by inhibiting function, and members of IGF family such as IGF-1, IGF-2, IGF-1R, IGF2R, and the like.

  Another group of cytotoxic or anti-tumor agents that can be used as effector molecules for component (B) of the transporter-load conjugate molecule of the present invention target tumor cell growth and tumor angiogenesis. May be selected from inhibitors. In this context, small molecule anti-tumor kinase inhibitors for malignant cells with anti-angiogenic activity and / or targets on vascular cells are particularly preferred. Kinase inhibitors such as EGFR, Her2 / neu, BCR-ABL, c-KIT, PKC, Raf, and PI3 inhibit angiogenesis by blocking secretion of angiogenic factors by diseased malignant cells. Kinase inhibitors such as VEGFR2, VEGFR1, PDGFR, PKC, Raf, and PI3 inhibit angiogenesis by acting on vascular cells. Examples of cyclin-dependent kinase synthesis inhibitors (CDKI) include olomoucine, flavopiridol, butyrolactone, and derivatives thereof, which limit the growth of tumor cells. On the other hand, as component (B) of the transporter-loading conjugate molecule of the present invention, suitable anti-tumor compounds may be selected from activators (eg, staurosporine) of cancer cell apoptosis programs, and anti-apoptotic proteins For example, Bcl-2 may be down-regulated.

  It is common to all of the above compounds that it must cross the cell membrane to act as an anticancer drug. Compounds belonging to each of these categories (compounds that directly damage DNA in the cell nucleus, compounds that act on the synthesis or disruption of mitotic spindles, or compounds that stop the synthesis of basic units of pre-DNA molecules) By binding to component (A) as component (B) to form the transporter-load conjugate molecule of the present invention, penetration of the anticancer compound into the cell is promoted and / or the solubility of the anticancer compound is increased. Improved the effectiveness of these therapeutic compounds. Consequently, the dosage of therapeutic anti-cancer compounds can be reduced by increasing cellular uptake and, preferably, improving the solubility of these compounds in an aqueous environment (eg, cytosol).

  Furthermore, component (B) of the transporter-loading conjugate molecule of the present invention may also comprise a small organic compound or drug molecule, such as a protease, in particular a protease, virus, bacterium or Protease inhibitors that inhibit protozoan proteases may also be included. In a preferred embodiment, these protease inhibitors (organic compounds or drug molecules) are used as part of the conjugate molecules of the present invention for viral infections, bacterial infections, or protozoal infections such as malaria. It is possible to have a function to treat. Specifically, viral infections such as retroviral diseases can be treated with protease inhibitors. The use of a conjugated molecule comprising a protease inhibitor is highly preferred for the treatment of HIV infection. Protease inhibitors used to bind to the carrier sequences disclosed herein include 640385, abacavir sulfate, AG1776, amprenavir (141W94 or VX-478), atazanavir (BMS-232632), cathepsin S protease inhibitor , D1927, D9120, Efavirenz, Emtricitabine, Enfuvirtide (T-20), Phosamprenavir (GW-433908 or VX-175), GS9005, GW640385 (VX-385), HCV protease inhibitor, Indinavir (MK-639) L-756, 423, Levopurin-ZG, lopinavir (ABT-378), lopinavir / ritonavir (LPV ABT-378 / r), MK-944A, mozenavir (DMP450), nelfinavir (AG-1343), nevirapine, P-1946, PL-100, purinostat, ritonavir (ABT-538), RO033-3649, TMC114, saquinavir (Ro-31-8959), tenofovir disoproxil fumarate, It may be selected from the group comprising tipranavir (PNU-140690), TLK19781, TMC-114, Vertex385, VX-950.

  Finally, effector molecules suitable as component (B) of the transporter-load conjugate molecule of the present invention are further separate from the label as defined above for the transporter-load conjugate molecule of the present invention. May be selected as a component. Such transporter-loading conjugate molecules of the invention are particularly suitable for in vitro or in vivo assays. In this situation, the label is a radioactive label, ie a radioactive phosphorylated label or a radioactive label containing sulfur, hydrogen, carbon, nitrogen, etc .; a colored dye (eg, digoxigenin); a fluorescent group (eg, fluorescein, rhodamine, below) A fluorescent chromoprotein as defined); a chemiluminescent group; or a combination of these labels. The fluorescent chromoprotein preferably comprises any fluorescent chromoprotein that can be activated to emit a fluorescent signal. More preferably, the fluorescent chromoprotein is selected from the group comprising any fluorescent chromoprotein, such as: green fluorescent protein (GFP); derivatives of green fluorescent protein (GFP), such as EGFP, AcGFP, TurboGFP, Emerald, thistle green, photoactivatable GFP (PA-GFP), etc .; blue fluorescent chromoprotein (BFP) including EBFP, sapphire, T-sapphire; high sensitivity cyan fluorescent protein (ECFP), mCFP, cerulean, CyPet Cyan fluorescent protein (CFP) containing; yellow fluorescent protein (YFP) containing topaz, venus, m citrine, Ypet, PhiYFP, m banana, yellow shifted green fluorescent protein (Yellow GFP), high sensitivity yellow fluorescent protein (EYFP); Kushibara orange, m orange, d tomato tandem, DsRed-monomer, m tangerine, m strawberry, monomeric red fluorescent protein (mRFP1) (also referred to herein as mRFP), m cherry, m raspberry, HcRed-tandem Selected from PA-GFP, CoralHue Dronpa (G), PS-CFP (C), PS-CFP (G), mEosFP (G), mEosFP (G) Optical highlighters; other monomeric fluorescent proteins such as kindling fluorescent protein (KFP1), aequorin, autofluorescent protein (AFP); or fluorescent proteins JRed, TurboGFP, PhiYFP, and PhiYFP-m, tHc-Red HcRed- Tandem), PS-CFP2, and KFP-Red (available from EVRΩGEN, www.evrogen.com see also), or other suitable fluorescent proteins.

  The transporter-load conjugate molecule of the present invention comprising components (A) and (B) may further comprise at least one optional additional component, preferably different from component (B). This at least one optional additional moiety can confer further function to the fusion protein of the invention. At least one optional additional component is a moiety (eg, HA, HSV-tag, His6-tag, FLAG-tag) that can facilitate purification and / or isolation of a transporter-loading conjugate molecule of the invention. There may be. Optionally, at the end of the production process, the components required for purification can be transferred to other transporter-load conjugate molecules of the invention (eg, by proteolytic cleavage, or other methods known in the art). May be removed from these components.

  Furthermore, any at least one additional component of the transporter-load conjugate molecule of the present invention may be identified, for example, preferably without losing the high cell permeability of the transporter-load conjugate molecule of the present invention. It may be a signal sequence or localization sequence that efficiently directs the transporter-loading conjugate molecule of the present invention to an intracellular target localization location or a specific cell type. In general, such signal sequences or localization sequences direct the transporter-loading conjugate molecules of the present invention to specific intracellular compartments, such as the endoplasmic reticulum, mitochondria, the Golgi apparatus, lysosomal vesicles, and the like. Lead. Exemplary signal or localization sequences include endoplasmic reticulum such as KDEL (SEQ ID NO: 218), DDEL (SEQ ID NO: 219), DEEL (SEQ ID NO: 220), QEDL (SEQ ID NO: 221), RDEL (SEQ ID NO: 222). Localized sequences; nuclear localization sequences such as PKKKKRKV (SEQ ID NO: 223), PQKKIKS (SEQ ID NO: 224), QPKKP (SEQ ID NO: 225), RKKR (SEQ ID NO: 226); ), Nuclear region localization sequences such as MPTRRRRPASASQALAPPTP (SEQ ID NO: 229); endosomal compartment localization sequences such as MDDQRDLISNNEQLP (SEQ ID NO: 230), but are not limited to these.

The transporter-loading conjugate molecules of the present invention may preferably further comprise at least one modification at the terminus, eg, either the C-terminus or the N-terminus, or both. The C-terminus may preferably be modified by amide modification, while the N-terminus may be modified by any suitable NH ₂ protecting group, such as acylation, or any of those already described for L-amino acids It may be modified by further modification.

  If components (A), (B), and further optional components (C), (D), and / or (E) of the transporter-load conjugate molecule of the present invention are present, these are also Generally, they are linked to each other via covalent bonds or electrostatic bonds (eg, polylysine), preferably via covalent bonds. In this context, the term “covalent bond” refers to a stable chemical bond between two atoms created by sharing one or more pairs of electrons. Preferably, when components (A) and (B) and further optional components (C), (D), and / or (E) of the transporter-loading conjugate molecule of the present invention are present, All of these can also be combined to form linear or non-linear (branched) molecules, preferably linear molecules. In a linear molecule, when the above components (A) and (B), and further optional components (C), (D), and / or (E) are present, all of these are also branched transporters— Without forming a cargo conjugate molecule, they are linked to each other through their ends in a linear form. Non-linear (branched) molecules, if any of the above components (A) and (B), and further optional components (C), (D), and / or (E) are present, are all For example, they are linked to each other via a terminus so as to be a branched transporter-loading conjugate molecule such as a Y-shaped form.

  Since component (A) of the transporter-loading conjugate molecule of the present invention is by definition a (poly) peptide sequence, the (covalent) linkage of additional component (B), as well as optional components (C), (D) , And / or (E), these (covalent) bonds are, of course, the type and nature of the components to be bound, ie one component is a protein, (poly) peptide, nucleic acid , (Small) organic compounds and the like.

  The order in which component (B) of the transporter-load conjugate molecule of the present invention binds to component (A) and to each other to form a linear molecule, as well as further optional components (C), (D), And / or (E), if present, the order in which they are combined with component (A) and with each other to form a linear molecule, generally may include any order. Thus, any of components (A), (B), and (C), (D), (E), etc., when present, can be bonded together. However, it is preferred that component (A) is attached to the terminus of the transporter-load conjugate molecule of the present invention. When component (B) is a protein or (poly) peptide sequence, and when components (C), (D), (E) are present, if any of these are protein or (poly) peptide sequences, Component (A) is the C-terminus of the transporter-loading conjugate molecule of the invention, for example when it occurs as a (poly) peptide or protein, component (B) C-terminus as defined above, or component (C), When (D), (E), etc. are present, it is preferably contained in these C-termini. The presence of the component (A) at such a position in the transporter-load conjugate molecule of the present invention means that the load (poly) peptide or protein sequence of the components (B), (C), (D), (E), etc. Are degraded by peptidases, particularly carboxypeptidases such as carboxy terminal peptidase N, before being transported to the desired target site, eg, cell, nucleus, etc. Alternatively, when the amino terminal peptidase is present in the cell line used, component (A) may be present at the amino terminus of the transporter-load conjugate molecule of the present invention.

  Transporter of the present invention when component (B) is a (poly) peptide or protein sequence and / or if any further optional components are present, these are (poly) peptide or protein sequences The bond between the protein or (poly) peptide component (A) of the cargo conjugate molecule and (B) and / or any further (poly) peptide component is preferably a (poly) peptide bond . Such (poly) peptide bonds may be formed using chemical synthesis involving both of the components to be bound (the N-terminus of one component and the C-terminus of the other component), or (poly) of both components It may be formed directly via protein synthesis of the entire peptide sequence, in which case it is preferred to synthesize both (protein or (poly) peptide) components in one step. Such protein synthesis methods include, for example, a liquid phase (poly) peptide synthesis method or a solid (poly) peptide synthesis method such as Merrifield's solid (poly) peptide method, t-Boc solid phase (poly) peptide synthesis, Fmoc solidification. Examples include, but are not limited to, phase (poly) peptide synthesis, solid phase (poly) peptide synthesis based on BOP (benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate), and the like.

  Furthermore, the component (A) and the component (B) of the transporter-loading conjugate molecule of the present invention can be bonded via a linker. When the component to be bonded has a reactive amino group or carboxy group, for example, It can also be linked directly (without a linker) by an amide bridge. Alternatively, an ester or ether bond is preferred.

  If the above mentioned further components (C), (D), and / or (E), etc. are present, these may be combined in a similar manner as component (A) and / or component (B), Or, optionally, bonded together and then bonded to either component (A) or component (B) as a single part. A linker sequence can also be used to fuse the transporter-load conjugate molecule of the present invention with at least one other component (see below). The method of attaching additional components to either component (A) or component (B) of the transporter-load conjugate molecule of the present invention depends on its chemical nature. If further components (C), (D), (E), etc. belong to the classification of peptide sequences, preferably the transporter-loading conjugate molecule of the invention binds at either end of component (A) Alternatively, for example, they are linked via the L-amino acid side chain or D-amino acid side chain of component (A) by a disulfide bond. Further components having other chemical properties can likewise be bound to component (A) (end groups or chemically active side groups) or component (B). The linkage via the side chain is preferably based on a side chain amino group, thiol group or hydroxyl group, for example via an amide bond, an ester bond or an ether bond. According to the present invention, all of the amino acids (any of components (C), (D), (E), etc., if composed of component (A) and amino acids) are all D-enantiomeric amino acids It should be noted that this ultimately reflects analogs that exist in nature by binding in a retro-inverso order. Nevertheless, when components (C), (D), (E), etc. are composed of amino acids, they may be composed of L-amino acids (in the sequence order that exists in nature), or D-amino acids and L -It may be composed of a combination with an amino acid.

  When a peptide linker sequence is used, the linker sequence preferably forms a mobile sequence of 2 to 10 residues, more preferably 1 to 5 residues. In a preferred embodiment, the linker sequence comprises at least 20%, more preferably at least 40%, even more preferably at least 50% Gly or β-alanine residues, such as GlyGlyGlyGlyGly, GlyGlyGlyGly, GlyGlyGly, CysGlyGly, or GlyGlyCly Including. One skilled in the art can readily select and prepare an appropriate linker sequence. A suitable linker sequence may be composed of D and / or L amino acids.

  The peptide linker sequence may also comprise component (A) of the transporter-loading conjugate molecule of the present invention, component (B) and / or further optional components, ie (C), (D), (E), etc. Wherein the amino terminal methionine is at least one of the following: component (A), and protein or (poly) peptide sequence components (B), (C), (D), ( E) is added before.

  Preferably, component (A) and component (B) are joined by chemical coupling by any suitable method known in the art, such as a crosslinking method. However, it should be noted that many chemical cross-linking methods are non-specific, i.e., the point of attachment is not limited to any particular site in the carrier or cargo portion. Therefore, the use of non-specific crosslinkers can attack functional sites or sterically block active sites to biologically infuse the fused components of the transporter-load conjugate molecules of the invention. There is a risk of activation. It is known to those skilled in the art to block groups that may react by using appropriate protecting groups. Alternatively, powerful and versatile oxime and hydrazone ligation techniques may be used, which are chemoselective entities that can be applied to the crosslinking of component (A) component (B). This cross-linking technique is described, for example, in Rose et al. (1994), JACS 116, 30. If further components (C), (D), (E), etc. are present, they are chemistry with each other or with component (A) and / or component (B) in a similar manner as described above. Can be coupled.

  By directing chemical coupling to functional groups that crosslink organic components of component (B) that are present only one or several in component (A), the specificity of the coupling can be increased. As an example, if there is only one cysteine residue in component (A) of the transporter-load conjugate molecule of the present invention, a cysteine thiol group can be used. Also, for example, when component (A) of the conjugate molecule does not contain a lysine residue, a cross-linking agent specific for the primary amine will be selective for the amino terminus of component (A). Alternatively, cross-linking can also be performed via the side chain of a glutamic acid residue located at the N-terminus of the (poly) peptide so that an amide bond can occur through the side chain. Therefore, it may be advantageous to attach a glutamic acid residue to the N-terminus of component (A) of the transporter-loading conjugate molecule of the present invention. However, when a cysteine residue is introduced into component (A), it is preferably introduced at or near the N-terminal or C-terminal. By adding one or more additional amino acids, for example, in particular cysteine residues, to the transport sequence or substituting at least one residue of the transport sequence contained in component (A). Conventional methods are available to change the amino acid sequence in this manner based on the modification. When a cysteine residue is used for coupling purposes, component (A) of the transporter-load conjugate molecule of the present invention preferably has one cysteine residue. The optional second cysteine residue should preferably not be present and can eventually be substituted when it occurs in component (A) of the transporter-loading conjugate molecule of the invention. When substituting cysteine residues in the original transport sequence used as component (A) or as part of component (A), it is generally necessary to change the folding structure of the (poly) peptide of component (A). It is desirable to keep it to a minimum. Changes in the folding structure of component (A) are minimized when cysteine is replaced with a chemically and sterically similar alternative. Accordingly, serine is preferred as an alternative to cysteine.

  The coupling of the two components of the transporter-load conjugate molecule of the present invention uses a coupling agent or conjugate agent that includes standard (poly) peptide synthesis coupling reagents such as HOBt, HBTU, DICI, TBTU, etc. Can be done. There are several intermolecular crosslinking reagents available, see, for example, Means and Feeney, Chemical Modification of Proteins, Holden-Day, 1974, pp. See 39-43. These reagents include, for example, N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) or N, N ′-(1,3-phenylene) bismaleimide; N, N′-ethylene-bis- (iodoacetamide) ), Or other such reagents having 6 to 11 carbon methylene bridges; and 1,5-difluoro-2,4-dinitrobenzene. Other crosslinking reagents useful for this purpose include p, p'-difluoro-m, m'-dinitrodiphenyl sulfone; dimethyl adipimidate; phenol-1,4-disulfonyl chloride; hexamethylene diisocyanate or Diisothiocyanate or azophenyl-p-diisocyanate; glutaraldehyde and disdiazobenzidine. The cross-linking reagent may be homobifunctional, i.e. have two functional groups that undergo the same reaction. A preferred homobifunctional cross-linking reagent is bismaleimidohexane (BMH). BMH contains two maleimide functional groups that react specifically with sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7). The two maleimide functional groups are linked by a hydrocarbon chain. Thus, BMH is useful for irreversible cross-linking of proteins (or polypeptides) that contain cysteine residues. The cross-linking reagent may be heterobifunctional. Heterobifunctional crosslinkers have two different functional groups, such as an amine reactive group and a thiol reactive group, which crosslink with two proteins each having a free amine and a thiol. Examples of heterobifunctional crosslinkers are succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and succinimide 4- ( p-maleimidophenyl) butyrate (SMPB), an extended chain analog of MBS. The succinimidyl group of these crosslinkers reacts with primary amines, and the thiol-reactive maleimide forms a covalent bond with the thiol of the cysteine residue. Since the crosslinking reagent often has low water solubility, a hydrophilic group such as a sulfonate group may be added to the crosslinking reagent to increase the water solubility. Sulfo-MBS and sulfo-SMCC are examples of cross-linking reagents with modified water solubility. Many cross-linking reagents yield conjugates that are essentially non-cleavable under cellular conditions. Thus, some cross-linking reagents contain covalent bonds such as disulfide bonds that are cleavable under cellular conditions. For example, Traut reagent, dithiobis (succinimidylpropionate) (DSP), and N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) are well-known cleavable crosslinkers. By using a cleavable cross-linking reagent, the components (B), (C), (D), (E), etc. of the cargo portion are delivered to the target cell and then the component (A) of the novel transporter construct Can be separated from For this purpose, direct disulfide bonds may be useful. Chemical cross-linking may also include the use of spacer arms. The spacer arm can provide intercellular mobility or adjust the intercellular distance between the conjugated moieties to help preserve biological activity. The spacer arm may be in the form of a protein (or polypeptide) moiety that includes spacer amino acids, eg, proline. Alternatively, the spacer arm may be part of a cross-linking reagent such as “long chain SPDP” (Pierce Chem. Co., Rockford, Ill., Catalog number 21651H). Many cross-linking reagents, including those discussed above, are commercially available. Detailed instructions for their use are readily available from commercial sources. A general reference for protein cross-linking and preparation of conjugates is Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press (1991).

  Those skilled in the art will be able to cup the different components in such a way that the different components of the transporter-loading conjugate molecule can transmit at least some of the individual spatial activities and / or properties throughout the transporter-loading conjugate molecule. Easily understand what should ring. For example, it is preferable that the coupling of the components does not completely lose leukocyte targeting.

  According to a further aspect, the present invention further provides a pharmaceutical composition, said pharmaceutical composition comprising a transporter-loading conjugate molecule of the present invention as defined above and optionally a pharmaceutically acceptable. Preferably, it comprises at least one of a possible carrier and a pharmaceutically acceptable vehicle, optional excipients, buffers, stabilizers, or other materials well known to those skilled in the art.

  The pharmaceutical composition of the invention comprises a transporter-load conjugate molecule of the invention as defined above. Preferably, said pharmaceutical composition comprises as component (A) at least one (poly) peptide targeting the WBC according to the invention and as component (B) a further substance (load). Component (B) may be any pharmaceutically active substance. Specific examples of such pharmaceutically active substances are disclosed above. Specifically, component (B) is involved in proteins or (poly) peptides, protein kinase inhibitors, in particular inhibitors of c-Jun amino terminal kinase, which is a protein kinase, antigens, antibodies, apoptotic factors, pathologies May be a therapeutically active effector molecule selected from a protease, preferably a peptide protease inhibitor, a BH3-domain BH3-only protein, and may be selected from nucleic acids, siRNA, cytotoxic agents, small organic compounds, etc. . As mentioned above, the transporter-loading conjugate molecule of the present invention may contain any additional components (C), (D), and / or (E).

As a further component, the pharmaceutical composition of the present invention may or may not contain a pharmaceutically acceptable carrier and / or vehicle. In the context of the present invention, a pharmaceutically acceptable carrier generally comprises a liquid or non-liquid base of the pharmaceutical composition of the present invention. When the pharmaceutical composition of the present invention is provided in liquid form, the carrier is generally a pyrogen-free water; isotonic saline or buffered (water) solution such as phosphate buffered solution, citrate buffered Such as a solution. In particular, when injecting the pharmaceutical composition of the present invention, a sodium salt, preferably at least 50 mmol / L (mM) sodium salt, a calcium salt, preferably at least 0.01 mmol / L (mM) calcium salt, and optionally Water or preferably a buffer, more preferably an aqueous buffer, containing a potassium salt, preferably at least 3 mmol / L (mM) potassium salt may be used. According to a preferred embodiment, the sodium salt, calcium salt and optionally potassium salt are in the form of their halides, such as chloride, iodide, or bromide, hydroxide, carbonate, bicarbonate, or sulfuric acid. It may be in the form of a salt or the like. Examples of sodium salts include, but are not limited to, NaCl, NaI, NaBr, Na ₂ CO ₃ , NaHCO ₃ , Na ₂ SO ₄ , and examples of optional potassium salts include KCl, KI , KBr, K ₂ CO ₃ , KHCO ₃ , K ₂ SO ₄ , and examples of calcium salts include CaCl ₂ , CaI ₂ , CaBr ₂ , CaCO ₃ , CaSO ₄ , and Ca (OH) _2. . Furthermore, the organic anion of the above-mentioned cation may be contained in the buffer. According to a more preferred embodiment, a buffer suitable for injection purposes as defined above contains a salt selected from sodium chloride (NaCl), calcium chloride (CaCl ₂ ), and optionally potassium chloride (KCl). Here, further anions may be present in addition to the chloride. In addition, CaCl ₂ may be replaced with another salt such as KCl. Generally, the salt in the injection buffer is at least 50 mmol / L (mM) sodium chloride (NaCl), at least 3 mmol / L (mM) potassium chloride (KCl), and at least 0.01 mmol / L (mM). ) At a concentration of calcium chloride (CaCl ₂ ). Injectable buffers may be hypertonic, isotonic, or hypotonic compared to a specific reference medium, i.e., the buffer has a greater, equal or less salt content than a specific reference medium. Preferably, salt concentrations as described above that do not damage cells by osmosis or other concentration effects can be used. The reference medium is a liquid that can be used as a reference medium in an “in vitro” method such as, for example, blood, lymph, cytoplasmic fluid, or other body fluid, or in an “in vitro” method such as a general buffer or liquid. It is. Such common buffers or liquids are known to those skilled in the art. Ringer's lactate solution is particularly preferred as the liquid base.

  However, one or more compatible solid or liquid fillers, diluents, or encapsulating compounds suitable for administration to the patient being treated can be used for the pharmaceutical compositions of the invention as well. As used herein, the term “compatibility” refers to a method in which the invention does not occur in an interaction that does not substantially reduce the pharmacological effectiveness of the pharmaceutical composition of the invention under typical use conditions. Means that these components of the pharmaceutical composition can be mixed with the transporter-loading conjugate molecules of the invention as defined above. Pharmaceutically acceptable carriers, fillers, and diluents, of course, must be sufficiently pure and low in toxicity to be in a state suitable for administration to the human being treated. Pharmaceutically acceptable carriers, fillers, or compounds that can be used as these components include, for example, sugars such as lactose, glucose, and sucrose; starches such as corn starch or potato starch; Cellulose, such as ethyl cellulose, cellulose acetate and derivatives thereof; powdered tragacanth; malt; gelatin; tallow; solid lubricants such as stearic acid, magnesium stearate, calcium sulfate; Vegetable oils such as olive oil, corn oil, and cacao-derived oils; for example, polyols such as polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; It is an acid.

  The pharmaceutical composition of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, via an implantable container, or any other known to those skilled in the art. It can be administered by any suitable route. As used herein, the term parenteral means subcutaneous, intravenous, intramuscular, intraarticular, bursa, intramatrix, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal Including intrapulmonary, intraperitoneal, intracarpal, intraarterial, and sublingual injection or infusion techniques.

  Preferably, the pharmaceutical composition of the present invention can be administered by parenteral injection, more preferably subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intramatrix, intrathecal, intrahepatic, Administration is by intralesional, intracranial, transdermal, intradermal, intrapulmonary, intraperitoneal, intracarpal, intraarterial, and sublingual injection or via infusion techniques. Sterile injectable forms of the pharmaceutical compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as a solvent or suspending solvent. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are also useful in injectable preparations, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially these polyoxyethylated forms. These oil solutions or suspensions are also long chain alcohol diluents or dispersants, such as carboxymethylcellulose commonly used in formulations in pharmaceutically acceptable dosage forms, including emulsions and suspensions. A similar dispersant may be contained. Also commonly used in the manufacture of other commonly used surfactants such as Tween, Span, and other emulsifiers, or pharmaceutically acceptable solids, liquids, or other dosage forms. Bioavailability enhancers may be used to formulate the pharmaceutical compositions of the present invention.

  For intravenous, dermal, or subcutaneous injection, or injection into the affected area, the active ingredient is in the form of a parenterally acceptable aqueous solution that does not contain pyrogens and has suitable pH, isotonicity, and stability. Preferably there is. A person skilled in the art can prepare a suitable solution using isotonic vehicles such as sodium chloride injection, Ringer's injection, and lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may optionally be included.

  The pharmaceutical composition of the invention as defined above may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions, or solutions. Good. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricants such as magnesium stearate are also commonly added. When administered orally in capsule form, useful diluents include lactose and dried corn starch. When an aqueous suspension is required for oral administration, the active ingredient, ie the transporter-loading conjugate molecule of the invention as defined above, is combined with emulsifiers and suspending agents. If necessary, specific sweetening, flavoring, or coloring agents may be added.

  Also, the pharmaceutical composition of the present invention is topical, particularly when the target of treatment comprises a region or organ that is readily accessible by topical application including, for example, skin diseases or diseases of any other accessible epithelial tissue. It may be administered. Suitable topical formulations are readily prepared for each of these areas or organs. When applied topically, the pharmaceutical composition of the invention comprises an immunostimulatory composition of the invention suspended in or dissolved in one or more carriers, in particular a suitable ointment containing the components defined above. Can be formulated. Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition of the present invention can be formulated into a suitable lotion or cream. In the context of the present invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Not.

  A “prophylactically effective amount” whether administered to an individual as a component (B) is a (poly) peptide, a nucleic acid molecule, or any other pharmaceutically useful compound according to the present invention. Or it is preferably administered in a “therapeutically effective amount” (in some cases), which is an amount sufficient to show a beneficial effect on the individual. The actual dosage, rate of administration, and time course of administration will depend on the nature and severity of the individual being treated.

  Since the (poly) peptide targeting WBC used in the present invention is incorporated in a large amount by cells, particularly WBC, the transporter-loading conjugate molecule (active ingredient) in the pharmaceutical composition administered to the subject The amount is not limited, but may be a very low dose. Thus, the dose may be much less than peptide drugs known in the art, such as DTS-108 (Florence Meyer-Losic et al., Clin Cancer Res., 2008, 2145-53). This provides a number of beneficial aspects such as, for example, reducing side reactions that may occur and reducing costs.

  The dose (per kg body weight) is preferably 10 mmol / kg or less, more preferably 1 mmol / kg or less, still more preferably 100 μmol / kg or less, still more preferably 10 μmol / kg or less, particularly preferably 1 μmol / kg or less. Particularly preferred is 100 nmol / kg or less, and most preferred is 50 nmol / kg or less.

  Accordingly, the dosage ranges are preferably about 1 pmol / kg to about 1 mmol / kg, about 10 pmol / kg to about 0.1 mmol / kg, about 10 pmol / kg to about 0.01 mmol / kg, about 50 pmol / kg to about 1 μmol / kg. kg, about 100 pmol / kg to about 500 nmol / kg, about 200 pmol / kg to about 300 nmol / kg, about 300 pmol / kg to about 100 nmol / kg, about 500 pmol / kg to about 50 nmol / kg, about 750 pmol / kg to about 30 nmol / kg kg, about 250 pmol / kg to about 5 nmol / kg, about 1 nmol / kg to about 10 nmol / kg, or a combination of any two of the above values.

  Those skilled in the art will recognize that the effective amount of the transporter-loading conjugate molecule according to the present invention is not only positively influenced by the advantageous properties of the (poly) peptide targeting the WBC according to the present invention, It is understood that it also depends on the effectiveness of the drug or effector molecule conjugated to the (poly) peptide targeting WBC. Thus, the actual preferred dose may vary depending on the transporter-load conjugate molecule.

  In this situation, treatment instructions, such as dose determination, are generally within the responsibility of general practitioners and other physicians when using the pharmaceutical compositions, and generally treatment Considerations, individual patient status, delivery site, mode of administration, and other factors known to the practitioner are considered. Examples of the techniques and protocols described above can be found in REMINGTON'S PHARAMEUTICAL SCIENCES, 16th edition, Osol, A.I. (Ed), 1980. Accordingly, the pharmaceutical compositions of the present invention generally comprise a “safe and effective amount” of the components of the pharmaceutical composition of the present invention, particularly the transporter-load conjugate molecules of the present invention as defined above. As used herein, a “safe and effective amount” is a transport of the invention as defined above that is sufficient to significantly induce a beneficial change in a disease or disorder as defined herein. By amount of body-load conjugate molecule. At the same time, however, a “safe and effective amount” is small enough to avoid serious side effects, ie, allows a reasonable relationship between benefits and risks. The determination of these limits is generally within reasonable medical judgment. The “safe and effective amount” of the components of the pharmaceutical composition of the invention, in particular the transporter-loading conjugate molecule of the invention as defined above, will further vary in relation to the particular condition being treated, and Within the knowledge and experience of the attending physician, the age and physical condition of the patient being treated, weight, general health, sex, diet, time of administration, elimination rate, combination of drugs, books defined above Activity, severity of symptoms, duration of treatment, combination of specific components (A), (B), (C), (D), and / or (E) of the transporter-load conjugate molecule of the invention It will also vary according to the nature of the treatment, the nature of the particular pharmaceutically acceptable carrier employed, and similar factors. The pharmaceutical composition of the present invention can generally be used as a pharmaceutical composition or vaccine for humans and for veterinary purposes, preferably for medical purposes for humans.

  According to a particular embodiment, for example, component (B) of the transporter-loading conjugate molecule of the invention is a (protein or (poly) peptide) antigen or antibody fragment suitable for eliciting an immune response, or In the case of a therapeutically active protein such as any of the above molecules, the pharmaceutical composition of the present invention may be provided as a vaccine. Such a vaccine of the present invention generally comprises the pharmaceutical composition of the present invention and the like, and the components (B), (C), (D) of the transporter-loading conjugate molecule of the present invention as defined above. Depending on the nature, such as (E), and / or, it is preferable to support the innate and / or adaptive immune response of the immune system of the patient being treated. An example of this is that if any of these components provides or encodes a ((poly) peptide) antigen or antibody fragment, the vaccine generally leads to an adaptive immune response in the patient being treated. It is done. Similarly, any of the additional components (B), (C), (D), and / or (E), etc., of the transporter-loading conjugate molecules of the invention as defined herein are innate immunity Responses and / or adaptive immune responses can be induced.

  The vaccine of the present invention may also comprise a pharmaceutically acceptable carrier, adjuvant, and / or vehicle as defined above for the pharmaceutical composition of the present invention. In the particular context of the vaccine of the present invention, the selection of a pharmaceutically acceptable carrier is largely determined by the method by which the vaccine of the present invention is administered. The vaccine of the present invention can be administered, for example, systemically or locally. In general, systemic routes of administration include, for example, transdermal, oral, parenteral routes, which include subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injection, and intranasal administration. Includes at least one of the routes. In general, topical routes of administration include, for example, topical routes of administration, but also include intradermal, transdermal, subcutaneous, or intramuscular injection, or intralesional, intracranial, intrapulmonary, intracarpal, and sublingual injections. Including. More preferably, the vaccine can be administered by the intradermal, subcutaneous or intramuscular route. Accordingly, the vaccine of the present invention is preferably formulated in a liquid (or sometimes solid) form. The appropriate amount of the vaccine of the invention to be administered can be determined by routine experimentation using animal models. Such models include, but are not limited to, rabbits, sheep, mice, rats, dogs, and non-human primate models. Preferred unit dosage forms for injection include sterile solutions of water, physiological saline, or mixtures thereof. The pH of such a solution should be adjusted to about 7.4. Preferred carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid, and collagen matrices. Pharmaceutically acceptable carriers suitable for topical application include those suitable for use in lotions, creams, gels and the like. When the vaccine of the present invention is administered orally, tablets, capsules and the like are preferred unit dosage forms. Pharmaceutically acceptable carriers for preparing unit dosage forms that can be used for oral administration are well known from the prior art. The choice depends on secondary considerations such as taste, cost, and shelf life that are not critical to the purpose of the present invention and can be readily made by one skilled in the art.

  The vaccine of the present invention may further contain one or more auxiliary substances in order to further enhance immunogenicity. Thereby, it is preferred to obtain a synergistic effect between the transporter-loading conjugate molecule of the invention as defined above and an auxiliary substance that may optionally be contained in the vaccine of the invention. Depending on the different types of auxiliary substances, different mechanisms can be considered. For example, compounds capable of maturing dendritic cells (DC), such as lipopolysaccharide, TNF-α or CD40 ligand, form the first class of suitable auxiliary substances. Generally, immunity, such as “danger signals” (LPS, GP96, etc.) or cytokines such as GM-CFS, which can targetically enhance and / or influence the immune response produced by the immunostimulatory adjuvant according to the invention. Any agent that affects the system can be used as an auxiliary substance. Particularly preferred auxiliary substances are cytokines such as monokines, lymphokines, interleukins or chemokines that further promote the innate immune response, for example IL-1, IL-2, IL-3, IL-4, IL-5. , IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL -19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31 Growth factors such as IL-32, IL-33, INF-α, IFN-β, INF-γ, GM-CSF, G-CSF, M-CSF, LT-β or TNF-α, hGH, etc. .

  Further additives that may be included in the pharmaceutical composition or the vaccine of the present invention include, for example, emulsifiers such as Tween®; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; pharmaceutical carriers; Agents; stabilizers; antioxidants; preservatives and the like.

  The vaccine of the present invention may also be produced by binding affinity (as a ligand) to the human Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10 or the mouse Toll-like receptor TLR1. Any further known to be immunostimulatory by binding affinity (as a ligand) to TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, or TLR13 A compound may be further contained.

  Another class of compounds that can be added to the vaccines of the invention in this context may be CpG nucleic acids, in particular CpG-RNA or CpG-DNA. CpG-RNA or CpG-DNA is single-stranded CpG-DNA (ssCpG-DNA), double-stranded CpG-DNA (dsDNA), single-stranded CpG-RNA (ssCpG-RNA), or double-stranded CpG-RNA. (DsCpG-RNA). The CpG nucleic acid is preferably in the form of CpG-RNA, and more preferably in the form of single-stranded CpG-RNA (ssCpG-RNA). Preferably, the CpG nucleic acid comprises at least one or more (mitogenic) cytosine / guanine dinucleotide sequences (CpG motif). According to the first preferred other embodiment, at least one CpG motif contained in these sequences, that is, C (cytosine) and G (guanine) of the CpG motif are not methylated. Optionally, all further cytosine or guanine included in these sequences may be methylated or unmethylated. However, according to another preferred embodiment, the CpG motifs C (cytosine) and G (guanine) may also be present in methylated form.

  According to a further aspect of the present invention, the transporter-loading conjugate molecule of the present invention, the pharmaceutical composition of the present invention, or the vaccine of the present invention as defined above is any disease as defined herein. And can be used for the prevention, treatment, attenuation and / or amelioration of disorders (to prepare a pharmaceutical composition or vaccine as defined herein, preferably both).

  Specifically, using the transporter-loading conjugate molecule described in the present invention, the pharmaceutical composition of the present invention, or the vaccine of the present invention, a target substance (loading molecule) such as a drug and an effector molecule is efficiently converted into leukocytes. Targeted delivery. Thus, transport to leukocytes can be performed according to the present invention, for example, in vivo, in vitro, and / or ex vivo.

  As a result, a transporter-loading conjugate molecule as defined herein, a pharmaceutical composition of the invention, or a vaccine of the invention can be used to treat, prevent, attenuate, and / or treat diseases and / or disorders involving leukocytes. Or it can be used for recovery.

The term “disease and / or disorder involving leukocytes” as used herein refers to at least one of the following:
a) diseases and / or disorders caused by defects in the white blood cells themselves (defects in WBC are the main cause of the disease),
b) a disease and / or disorder where WBC deficiency is not a major cause of the disease but WBC contributes to the pathology and symptoms of said disease (WBC is a secondary cause of the disease),
c) WBC is neither a major or minor cause, nor does WBC have a detrimental effect, but by the action of leukocytes (which may be stimulated via the transporter-loading conjugate molecule of the present invention). A disease and / or disorder (and its symptoms) that can be treated, prevented, attenuated, or ameliorated.

  One non-exclusive and merely illustrative example of a) is a genetic defect of leukocytes, and WBC cancers such as leukemias and lymphomas. One non-exclusive and merely illustrative example of b) is many types of inflammatory diseases. One non-exclusive and merely illustrative example of c) is most infectious diseases.

  “Leukocyte” (WBC, also called white blood cell), as used herein, refers to any type of white blood cell. Leukocytes can be primary cells, immortalized cells, and / or transgenic cells. The leukocytes can be selected from, for example, granulocytes, lymphocytes, monocytes, macrophages, dendritic cells, mast cells, and / or microglia cells. The granulocytes can be selected from the group consisting of neutrophils, eosinophils, and basophils. The lymphocytes can be selected from, for example, NK cells, helper T cells, cytotoxic T cells, γδ T cells, and B cells. The term may also include various stages of development of progenitor cells and / or leukocytes.

  There are several options for using the transporter-loading conjugate molecule of the invention, the pharmaceutical composition of the invention, or the vaccine of the invention as defined above in a therapeutic method. For example, the transporter-loading conjugate molecules of the present invention are useful for the treatment, prevention, attenuation and / or recovery of diseases and / or disorders involving leukocytes selected from viral infections, particularly viral infections of leukocytes. Can be used.

  Tables 11 and 12 show examples of such viral infections, particularly leukocyte viral infections, and treatment examples (some can function as the component (B) of the present invention).

  Further examples of diseases and / or disorders involving leukocytes are shown in Tables 13-15.

  A transporter-loading conjugate molecule as defined above, a pharmaceutical composition of the invention, or a vaccine of the invention is used for the treatment, prevention, attenuation and / or recovery of a disease and / or disorder selected from: Preferably: cancer or tumor diseases including diseases caused by defective apoptosis, inflammatory diseases, infectious diseases including bacterial and viral (infectious) diseases, diseases strongly associated with JNK signaling, autoimmune disorders, Autoimmune disease, cardiovascular disease, neurological disease, neurodegenerative disease, liver disease, spinal disease, uterine disease, major depressive disorder, non-chronic inflammatory digestive system disease, chronic inflammatory digestive system disease, diabetes, hair loss, hearing loss Or inner ear disease. The transporter-loading conjugate molecules of the present invention may be used to treat transplanted organs / tissues / cells, or to treat recipient organs / tissues / cells, for example, to prevent leukocytes from initiating graft rejection. By treating the cells, it can also be used for tissue transplantation (to prepare a pharmaceutical composition).

  Prevention, treatment, attenuation and / or amelioration of a disease as defined herein generally includes administration of a pharmaceutical composition as defined above. The term “prevention” generally refers to preventing a disease as defined herein in a patient, preferably before the onset of the disease is manifested in the patient. The term “treatment” generally refers to when the disease may have already been diagnosed or is to be prevented, i.e., in parallel with onset before the onset of symptoms of the disease in the patient. And any treatment of a disease as defined herein in a patient after onset. For example, the term “treatment” as used in the term “treatment of symptoms” more preferably means administering at least a therapeutically effective amount of a therapeutic compound to elicit a therapeutic effect. It does not necessarily mean “cure”, but preferably means that it has at least some minimal physiological effects on such symptoms when administered to a living body having a symptom. For example, treatment can include administration of an agent and the presence of the agent that causes a physiological change in the recipient animal. “Attenuation” as used herein refers to the effect of slowing or stopping, for example, a particular stage, in the progression of other progressive diseases. Finally, the term “recovery” preferably includes any modulation of a disease as defined herein, preferably the positive modulation of a disease as defined herein. The particular modulation may depend on the disease being treated.

  A pharmaceutical composition, vaccine, or transporter-loading conjugate molecule of the invention as defined herein may be administered directly to a patient using the route of administration defined above for the pharmaceutical composition. it can. Alternatively, the pharmaceutical composition, vaccine, or transporter-loading conjugate molecule of the invention as defined above is an ex vivo approach, for example, a pharmaceutical composition, vaccine, or transporter of the invention as defined above- After introducing the cargo conjugate molecule into cells, preferably autologous cells, i.e. cells derived from the patient to be treated, and optionally storing and / or culturing these cells prior to treatment, the site of the patient to be treated. It may be administered to a patient by transplanting into the patient.

  According to one preferred embodiment, the transporter-loading conjugate molecule of the invention, the pharmaceutical composition of the invention, or the vaccine of the invention as defined above comprises, for example, a disease caused by defective apoptosis. Can be used to prevent, treat and / or remedy cancer or tumor diseases, wherein the diseases are preferably selected from: acoustic schwannomas, anal carcinomas, Astrocytoma, basal cell carcinoma, Behcet's syndrome, bladder cancer, blastoma, bone cancer, brain metastasis, brain tumor, brain cancer (glioblastoma), breast cancer (breast carcinoma), Burkitt lymphoma, carcinoid, cervical cancer, colon Carcinoma, colorectal cancer, internal body carcinoma, craniopharyngioma, CUP syndrome, endometrial carcinoma, gallbladder cancer, genital tumors including cancer of the genitourinary tract, glioblastoma, glioma, head / neck tumor, Hepatoma, Intestinal cancer, including histiocytic lymphoma, Hodgkin's syndrome or lymphoma, and non-Hodgkin's lymphoma, pituitary tumor, small intestine cancer tumor, gastrointestinal cancer, caposid's sarcoma, kidney cancer, renal carcinoma, laryngeal cancer, epigastric cancer; acute bone marrow Leukemia (AML), erythroleukemia, acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL); eyelid tumor, liver cancer, liver metastasis, lung carcinoma (= Lung cancer = bronchial carcinoma), small cell lung cancer and non-small cell lung carcinoma, and lung adenocarcinoma, lymphoma, lymph adenocarcinoma, malignant melanoma, breast carcinoma (= breast cancer), medulloblastoma, melanoma, meninges Tumor, mycosis fungoides, neoplastic disease sheath tumor, esophageal cancer, esophageal carcinoma (= esophageal cancer), oligodendroma, ovarian cancer (= ovarian carcinoma), ovarian carcinoma, pancreatic carcinoma (= pancreatic cancer), Penile cancer, penis cancer, pharyngeal cancer, pituitary tumor, trait Cystoma, prostate cancer (= prostate tumor), rectal carcinoma, rectal tumor, kidney cancer, renal carcinoma, retinoblastoma, sarcoma, Schneider's disease; basal cell and squamous cell carcinoma, and skin cancer including psoriasis, eg melanoma or non Melanoma skin cancer; pemphigus vulgaris, soft tissue tumor, squamous cell tumor, gastric cancer, testicular cancer, throat cancer, thymoma, thyroid carcinoma, tongue cancer, urethral cancer, uterine cancer, vaginal cancer; various virus-induced tumors, For example, papilloma virus-induced carcinoma (eg, cervical carcinoma = cervical cancer), adenocarcinoma, herpesvirus-induced tumors (eg, Burkitt lymphoma, EBV-induced B-cell lymphoma, cervical carcinoma), hepatitis B-induced Tumor (hepatocellular carcinoma), HTLV-1 and HTLV-2 induced lymphoma; vulvar cancer, wart condition or complications. In this context, the terms “therapy” and “therapeutic” preferably mean having at least some minimal physiological effects when administered to an organism. For example, the physiological effect of administering a “therapeutic” anti-tumor compound can be inhibition of tumor growth, or reduction in tumor size, or prevention of tumor recurrence. Preferably, in the treatment of cancer or neoplastic disease, compounds that inhibit tumor growth, reduce tumor size, or prevent tumor recurrence are considered therapeutically effective. Thus, the term “antitumor agent” preferably means any therapeutic agent that has a therapeutic effect on a tumor, neoplastic disease, or cancer.

  According to another preferred embodiment, the transporter-loading conjugate molecule of the invention, the pharmaceutical composition of the invention, or the vaccine of the invention as defined above is an inflammatory disease of the lung or an inflammation such as a lung disease. Can be used to prevent, treat, and / or ameliorate sexual illness, including: acute respiratory distress syndrome (ARDS), or pulmonary fibrosis; cystic Fibrotic tissue formation, including fibrosis, meningitis, and inflammation of tissues including but not limited to graft rejection or transplant rejection; asthma, chronic obstructive pulmonary disease (COPD), pneumonia, and pulmonary fibrosis Chronic diseases related to the respiratory system including:

  According to another preferred embodiment, the transporter-loading conjugate molecule of the invention, the pharmaceutical composition of the invention, or the vaccine of the invention as defined above is for example an infectious disease, preferably a virus, retro It can be used for the prevention, treatment, and / or recovery (preparation of drugs) of viral, bacterial, or parasitic infectious diseases. Such infectious diseases are generally selected from the following: AIDS, anthrax, Japanese encephalitis, bacterial infectious diseases such as miscarriage (prostate inflammation), anthrax, appendicitis, borreliosis, botulism, Campylobacter, trachoma chlamydia (Urethral inflammation, conjunctivitis), cholera, diphtheria, Donovan disease, typhoid fever, typhoid fever, gas gangrene, gonorrhea, barbarian fever, Helicobacter pylori, pertussis, inguinal lymph granuloma, osteomyelitis, legionellosis, fowlpox, cuspidus Condyloma, giant cell virus (CMV), dengue fever, early summer meningoencephalitis (ESME), Ebola virus, cold, fifth disease, foot-and-mouth disease, herpes simplex type I, herpes simplex type II, herpes zoster, HSV, parasite, protozoa Infectious diseases caused by animals or fungi, such as amoebiasis, schistosomiasis Bilharz, Chagas , Echinococcus, fish strepidae, fish poison (Siguatera), fox crested worms, chytrids, dogworms, candidiasis, yeast flora, scabies, cutaneous leishmaniasis, lambullous flagellum (giardia flagella) Worms), lice, malaria, microscopic examination, onchocercosis (ocular onchocercosis), fungal diseases, caterpillars, schistosomiasis, swine tapeworms, toxoplasmosis, trichomoniasis, trypanosomiasis (sleeping sickness), Visceral leishmaniasis, diaper rash / diaper dermatitis or scabies, infectious erythema, influenza, capo sarcoma, Lassa fever, leishmaniasis, leprosy, listeriosis, limeborreliosis, malaria, Marburg virus infection , Measles, meningitis including bacterial meningitis, molluscum contagiosum, mononucleosis, mumps, mycoplasma hominis, newborn Septicemia (chorionic amniitis), water cancer, Norwalk virus infection, otitis media, paratyphoid, Pfeiffer glandular fever, evil epidemic, pneumonia, polio (gray leukomyelitis, pediatric lameness), pseudocroup, rabies, Ritan syndrome, Rocky mountain erythema Fever, salmonella paratyphoid, salmonella typhoid, SARS, scarlet fever, shingles, hepatitis, pressure ulcer, flexible lower heel, syphilis, tetanus, three-day fever, tripper, tsutsugamushi disease, Mycobacterium tuberculosis, typhoid, vaginitis (vaginal) Inflammation), viral diseases caused by the following viruses, cytomegalovirus (CMV), orthopox variola virus, orthopox aristrim virus, sheep parapox virus, infectious molluscum virus, herpes simplex virus type 1 , Herpes simplex virus type 2, herpes B virus, chickenpox Herpes zoster virus, pseudorabies virus, human giant cell virus, human herpes virus type 6, human herpes virus type 7, Epstein-Barr virus, human herpes virus type 8, hepatitis B virus, chikungunya virus, onyonyon virus, ruby Virus, hepatitis C virus, GB virus C, West Nile virus, dengue virus, yellow fever virus, jumping disease virus, St. Louis encephalitis virus, Japanese B encephalitis virus, powasan virus, FSME virus, SARS, SARS-related coronavirus, Human coronavirus 229E, human coronavirus Oc43, torovirus, human T lymphocyte tropic virus type I, human T lymphocyte tropic virus type II, HIV (AIDS), ie human immunodeficiency virus type 1 or human immune Deficiency virus type 2, influenza virus, lassa virus, lymphocytic choriomeningitis virus, Tacaribe virus, Junin virus, Machupo virus, Borna disease virus, Bunyamwella virus, California encephalitis virus, Rift Valley fever virus, sand flies Fever virus, Tuscan virus, Crimea-Congo hemorrhagic fever virus, Hazara virus, Hasan virus, Hantan virus, Seoul virus, Prospect Hill virus, Pumara virus, Dobrava Belgrade virus, Tula virus, Sinnonbull virus, Lake Victoria Maru Burg virus, Zaire Ebola virus, Sudan Ebola virus, Côte d'Ivoire Ebola virus, Influenza virus type A, Influenza virus type B, Fluenza virus type C, parainfluenza virus, measles virus, mumps virus, RS virus, human metapneumovirus, vesicular stomatitis Indiana virus, rabies virus, mocola virus, dubenheige virus, European bat lyssa virus type 1 + 2 Australian bat lyssa virus, adenovirus type A-F, human papilloma virus, condyloma virus type 6, condyloma virus type 11, polyoma virus, adeno-associated virus type 2, rotavirus or orbivirus, chickenpox including varicella-zoster, and Virus infectious diseases such as malaria virus and AIDS, infectious diseases caused by warts, hollow warts, dengue fever, three-day fever, Ebola virus, cold, early summer meninges Encephalitis (FSME), flu, shingles, hepatitis, herpes simplex type I, herpes simplex type II, herpes simplex, influenza, Japanese encephalitis, Lassa fever, Marburg virus, warts, West Nile fever, yellow fever, etc.

  According to another preferred embodiment, the transporter-loading conjugate molecule of the present invention, the pharmaceutical composition of the present invention, or the vaccine of the present invention is used to prevent, treat, and treat diseases strongly associated with JNK signaling in a subject, and And / or can be used for recovery (preparing a drug for). Such a disease or disorder strongly associated with JNK signaling in a subject is preferably, but not limited to, selected from the following: autoimmune disorder, cardiovascular disease, cancer or tumor disease as defined above, 1 Diabetes including type 2 diabetes and type 2 diabetes, inflammatory diseases as defined above, hair loss including alopecia areata, lung disease, neurological disease, neurodegenerative disease, liver disease, spinal disease, uterine disease, (viral) infection Sexual disorders and depressive disorders. In the case of a disease or disorder strongly associated with JNK signaling, the term “recovery” refers to suppressing JNK expression when JNK is overexpressed, and / or c-jun in any of the above diseases, Suppressing phosphorylation of ATF2 or NFAT4, which may be, for example, in the cell as a competitive inhibitor of natural c-jun, ATF2, and NFAT4 binding sites within the above definition. This is done by using at least one JNK inhibitor sequence as defined herein that is bound to a novel transporter molecule. In this particular context, the term “regulation” includes, but is not limited to, hetero- and homo-complexes of transcription factors composed of c-jun, ATF2, or NFAT4, and their associated partners, eg, c It also includes inhibiting the AP-1 complex composed of -jun, AFT2, and c-fos. Such suppressive JNK inhibitor sequences may be introduced into cells when a disease or disorder strongly associated with JNK signaling as defined above is associated with overexpression of JNK. In some cases, “modulation” in the context of a disease or disorder strongly associated with JNK signaling is IB-related using, for example, IB (poly) peptide specific antibodies that block the binding of IB-peptides to JNK. Increasing JNK expression by blocking JNK inhibition by (poly) peptides may be included. Prevention and / or treatment of a subject with a pharmaceutical composition as disclosed above is generally accomplished by administering to the subject an (in vivo) (“therapeutically effective”) amount of the pharmaceutical composition. The subject can be, for example, any mammal, such as a human, primate, mouse, rat, dog, cat, cow, horse, or pig. The term “therapeutically effective” means that the active ingredient of the pharmaceutical composition is in an amount sufficient to ameliorate a disease or disorder strongly associated with JNK signaling as defined above. Additional examples can be found for other diseases mentioned herein.

  Accordingly, the transporter-loading conjugate molecule of the present invention, the pharmaceutical composition of the present invention, or the vaccine of the present invention provides (preparation of a drug) for prevention, treatment, and / or amelioration of an autoimmune disorder or disease. Can be used for Autoimmune disorders or diseases can be broadly classified into systemic autoimmune disorders and organ-specific, ie localized autoimmune disorders, depending on the main clinicopathological features of each disease. Autoimmune diseases that can be classified into systemic syndromes include systemic lupus erythematosus (SLE), Sjogren's syndrome, scleroderma, rheumatoid arthritis and polymyositis, or endocrine (type 1 diabetes (type 1 diabetes mellitus). ), Hashimoto's thyroiditis, Addison's disease, etc.), cutaneous (acne vulgaris), blood (autoimmune hemolytic anemia), neurological (multiple sclerosis), or localized syndrome, or In some cases, it may include virtually any localized mass of body tissue. The autoimmune disease to be treated can be selected from the group consisting of: type I autoimmune disease, type II autoimmune disease, type III autoimmune disease, or type IV autoimmune disease, eg, multiple sclerosis (MS), rheumatoid arthritis, diabetes, type I diabetes mellitus (type 1 diabetes mellitus), chronic polyarthritis, basedo disease, autoimmune form of chronic hepatitis, ulcerative colitis, type I allergic disease, type II allergic disease, III Type allergic disease, type IV allergic disease, fibromyalgia, alopecia, Bechteref disease, Crohn's disease, myasthenia gravis, chronic simple lichen, rheumatic polymyalgia, progressive systemic sclerosis (PSS), Reitan syndrome, rheumatoid arthritis, psoriasis, vasculitis, etc., or type II diabetes. Although the exact mechanism for why the immune system induces an immune response to self-antigens has not yet been clarified, there are some findings regarding the pathogenesis. According to it, the self-reaction may be due to T cell bypass. The normal immune system requires activation of B cells by T cells, and after activation, B cells can produce antibodies in large quantities. This T cell requirement may be rarely bypassed, for example by direct binding of the β-subunit to the T cell receptor non-specifically, to the B cell, or even the T cell. For example, when an organism producing a superantigen capable of initiating polyclonal activation is infected. Another interpretation speculates that the autoimmune disease may be due to molecular mimicry. Since foreign antigens may share structural similarities with a particular host antigen, any antibody produced against this antigen (which mimics a self-antigen) is theoretically a host antigen. May amplify the immune response. The most prominent form of molecular mimicry is found in group A β-hemolytic streptococci, which shares antigen with the human myocardium and is involved in symptomatic expression in the heart of rheumatic fever.

  The transporter-loading conjugate molecule of the present invention, the pharmaceutical composition of the present invention, or the vaccine of the present invention is used for the prevention, treatment and / or recovery (preparation of a drug) for cardiovascular disease. The disease is preferably selected from heart disease and coronary heart disease, arteriosclerosis, stroke, abdominal aortic enlargement such as aortic hypertension, and myocardial infarction.

  Further, the transporter-loading conjugate molecule of the present invention, the pharmaceutical composition of the present invention, or the vaccine of the present invention can be used to prepare a drug for the prevention, treatment and / or recovery of a neurological or neurodegenerative disease. The disease may include, but is not limited to, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), ataxia, epilepsy; glaucoma, eye including eye infection Neurological disease; multiple sclerosis, meningitis; “dissection” or breakage of axons, eg neurological diseases caused by disorders or diseases of the nervous system, including axotomy; pain, in particular neuropathic pain; Selected from stroke, including bloody stroke; and viral encephalopathy.

  The transporter-loading conjugate molecule of the present invention, the pharmaceutical composition of the present invention, or the vaccine of the present invention includes, but is not limited to, prevention, treatment, and / or liver disease selected from hepatitis and hepatotoxicity. It can also be used for recovery (preparing drugs).

  Further, the transporter-loading conjugate molecule of the present invention, the pharmaceutical composition of the present invention, or the vaccine of the present invention includes, but is not limited to, prevention, treatment, and / or spinal disease selected from intervertebral hernia. It can also be used for recovery (preparing drugs).

  According to one preferred embodiment, the transporter-loading conjugate molecule of the present invention, the pharmaceutical composition of the present invention, or the vaccine of the present invention includes, but is not limited to, a uterus selected from endometriosis. It can also be used for the prevention, treatment and / or amelioration (preparation of drugs) of diseases.

  According to another preferred embodiment, the transporter-loading conjugate molecule of the present invention, the pharmaceutical composition of the present invention, or the vaccine of the present invention is not limited, but is also known as major depression. Depressive disorder, unipolar depression, clinical depression, ie prevention, treatment and / or recovery of (depressing) a depressive disorder selected from mere depression, bipolar disorder, mania, and manic depression Can also be used.

  According to a further preferred embodiment, the transporter-loading conjugate molecule of the present invention, the pharmaceutical composition of the present invention, or the vaccine of the present invention is used for the prevention, treatment of non-chronic or chronic inflammatory gastrointestinal diseases in a subject, And / or can be used for recovery (preparing a drug for). The term “non-chronic or chronic inflammatory gastrointestinal disease” as used herein generally refers to a non-chronic or chronic inflammatory disease associated with the gastrointestinal tract. This includes the esophagus, stomach, duodenum first part, second part, third part and fourth part, jejunum, ileum, ileocecum, large intestine, (ascending colon, transverse colon, and descending colon) sigmoid colon And rectal diseases. In this regard, it is also preferable to include chronic inflammatory gastrointestinal diseases characterized by inflammation of the large intestine such as colitis. Examples of the diseases include ulcerative colitis (ulcerative colitis), Crohn's disease (Crohn's disease) ), Free colitis, ischemic colitis, infectious colitis, fulminant colitis, drug-induced colitis, microscopic colitis, lymphocytic colitis, collagen colitis, atypical colitis, and atypical colitis A flame etc. are mentioned.

  In the above situation, the present invention also provides a transporter-loading conjugate molecule of the present invention, a pharmaceutical composition of the present invention for preventing, treating and / or ameliorating a disease or disorder referred to in the present invention, Or it relates to the use of the vaccine according to the invention. The invention also includes the use of these components, particularly for inoculating or using the transporter-loading conjugate molecule of the invention, the pharmaceutical composition of the invention, or the vaccine of the invention as an inoculum. According to a particularly preferred embodiment of the present invention, a method for preventing, treating and / or ameliorating the above mentioned diseases or disorders or an inoculation method for preventing the above mentioned diseases is generally described in the above book. A patient in need of the transporter-loading conjugate molecule, pharmaceutical composition, or vaccine of the invention (eg, a patient suffering from or exhibiting a symptom of the disease), In particular to humans, preferably administered in a “safe and effective amount” by one of the above formulations as described above. Also, the method of administration may be as described above for the pharmaceutical composition or vaccine of the present invention.

  According to a fifth aspect of the invention, targeting the pharmaceutical composition of the invention as defined above, the vaccine of the invention, the transporter-loading conjugate molecule of the invention, the WBC as defined herein. The (poly) peptide, or these variants or fragments within the above definition can be used as a drug. Such an agent may be a pharmaceutical composition or a vaccine as indicated above. They can be utilized in medical applications in general, preferably for any of the prevention, treatment and / or recovery of the diseases or disorders mentioned herein.

  According to a further aspect of the invention, the transporter-load conjugate molecule of the invention is utilized to transport any cargo molecule (preferably as defined herein) to the leukocytes of the patient to be treated. can do. In this situation, the cargo molecule may be suitable for the therapy referred to herein, particularly the prevention, treatment and / or amelioration of the disease or disorder referred to herein, and the cargo molecule is From any suitable cargo molecule, more preferably any of the components (B), (C), (D), and / or (E) etc. of the transporter-load conjugate molecules of the invention It may be selected from any of the cargo molecules described.

  In one embodiment of the invention, the transporter-loading conjugate molecule as defined above, the pharmaceutical composition of the invention, or the vaccine of the invention is one, two or more diseases selected from: And / or not used for treatment, prevention, attenuation and / or recovery of disorders: cancer or tumor diseases including diseases caused by defective apoptosis, inflammatory diseases, viral (infectious) diseases, JNK signals Diseases strongly related to transmission, autoimmune disorder, autoimmune disease, cardiovascular disease, neurological disease, neurodegenerative disease, liver disease, spinal disease, uterine disease, major depressive disorder, non-chronic inflammatory digestive system disease, chronic inflammation Sexual digestive disorders, diabetes, and / or hair loss. Rather, they are selected from the remaining diseases and / or disorders disclosed herein. Similarly, in one embodiment of the invention, the transporter-loading conjugate molecule described herein does not include a JNK inhibitor, but includes other agents disclosed herein. In a further embodiment of the invention, the transporter-loading molecule described herein does not comprise SEQ ID NO: 1 as component (A), but for example one of the examples disclosed herein Another component (A) such as one.

  According to a further aspect of the invention, the (poly) peptide targeting WBC, the transporter-loading conjugate molecule of the invention as defined above, or a variant or fragment thereof within the above definition, the invention The pharmaceutical composition of the present invention, or the vaccine of the present invention, can be used in diagnostics, for example (in vivo or in vitro) assays, as a diagnostic tool for detecting, predicting, diagnosing, or monitoring various symptoms and conditions of the mentioned disorder or disease. For example, in an immunoassay.

  As an example, an immunoassay can be performed by a method comprising contacting a patient-derived sample with a transporter-load conjugate molecule of the present invention as defined above, wherein the transporter-load of the present invention. Component (B) of the conjugate molecule, and / or at least one of components (C), (D), and / or (E) is a component or compound contained in the sample, such as (cell) Specific components or compounds may be targeted. Any of such components (B) or components (C), (D), and / or (E) of the transporter-load conjugate molecule of the present invention may be, for example, a (cell) specific component or compound of a sample Wherein such (cell) specific component or compound of the sample is, for example, component (B), (C), (D), and / or (E) as defined herein. ) May be a compound or component as described above. Contacting the sample is generally performed under conditions that allow immunospecific binding to occur and then detect or measure the amount of any immunospecific binding by the antibody. In certain embodiments, specific for a (cell) specific component or compound of a sample, such as component (B), (C), (D), and / or (E), etc. as defined herein. A tissue or serum sample from a patient for the presence of components (B), (C), (D), and / or (E) as defined above, or for diseases associated therewith Can be analyzed. Available immunoassays include Western blot, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), “sandwich” immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, aggregation assay, fluorescence immunoassay. Competitive and non-competitive assay systems using techniques such as, but not limited to, complement binding assays, immunoradiometric assays, and protein-A immunoassays.

  Alternatively, (in vitro) assays generally involve a pharmaceutical composition, vaccine, or transporter-loading conjugate molecule of the invention as defined above, or a variant or fragment thereof within the above definition. Can be performed, for example, by delivering to a target cell selected from cultured animal cells, human cells, or microorganisms and monitoring the response of the cells by biophysical methods generally known to those skilled in the art. The target cells generally used herein may be cultured cells (in vitro) or in vivo cells, ie cells containing a living animal or human organ or tissue, or a microorganism found in a living animal or human. . Particularly preferred in this context are so-called markers or labels, which are components (B) or components (C), (D) and / or (E) of the transporter-loading conjugate molecules of the invention. And such label may be a label as generally defined above for the transporter-load conjugate molecules of the invention.

  In a further aspect, the invention relates to the use of a (poly) peptide targeting the WBC according to the invention in the production of a transporter-loading conjugate molecule for transporting a substance of interest (a cargo molecule) to a leukocyte.

In a further embodiment, the present invention also provides a method for transporting a target substance (a cargo molecule) to a white blood cell, comprising:
i) contacting a transporter-loading conjugate molecule with a white blood cell, wherein the transporter-loading conjugate molecule comprises:
a) As a component (A), an amino acid sequence fragment represented by HIV TAT protein (SEQ ID NO: 1), a variant, or a (poly) peptide comprising a variant of said fragment
b) as component (B), a cargo molecule, and c) one or more further optional components,
Relates to a method comprising:

The method may be a treatment method, i.e. contact performed in the subject to be treated. Alternatively, the method may be an ex vivo method or an in vitro method. Accordingly, the present invention, in a further embodiment, is an (isolated) leukocyte comprising a transporter-load conjugate molecule, wherein the transporter-load conjugate molecule comprises:
a) As a component (A), an amino acid sequence fragment represented by HIV TAT protein (SEQ ID NO: 1), a variant, or a (poly) peptide comprising a variant of said fragment
b) as component (B), a cargo molecule, and c) one or more further optional components,
Relates to leukocytes including

  The present invention also relates to leukocytes comprising only the remaining fragments of the transporter-loading conjugate molecule. This may be the case when the transporter-load conjugate molecule includes, for example, a protease cleavage site that leads to degradation of the original transporter-load conjugate molecule.

  According to the last aspect of the present invention, the present invention also provides (poly) peptides targeting WBC or fragments or variants thereof, transporter-loading conjugate molecules of the present invention, pharmaceutical compositions of the present invention. And / or kits, in particular parts of the kits, containing technical instructions containing the vaccines of the present invention alone or in combination as components and optionally containing information on the administration and dosing of these components. Such a kit, in particular a part of the kit, can be applied, for example, in the uses or uses described above. The present invention more particularly provides for the use of a kit for diagnostic or therapeutic purposes, in particular for the treatment, prevention or monitoring of a disclosed disease or disorder.

  The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, in addition to the embodiments described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are within the scope of the appended claims.

  Various publications are cited herein, the disclosures of which are hereby incorporated by reference in their entirety.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

(Example 1: Preparation of JNK inhibitor fusion protein)
The C-terminus of SEQ ID NO: 121 is derived from HIV-TAT4g 57 according to SEQ ID NO: 3 (Vives et al., J Biol. Chem. 272: 16010 (1997)) via a linker consisting of two proline residues. A JNK inhibitor fusion protein according to SEQ ID NO: 234 (L-TAT-IB1 (s)) was synthesized by covalently binding to an N-terminal 10 amino acid carrier peptide. This linker was used to maximize mobility and prevent unwanted secondary structure changes. Basic structures were also prepared and named L-IB1 (s) (SEQ ID NO: 121) and L-TAT (SEQ ID NO: 3), respectively.

  The all-D-retroinverso peptide according to SEQ ID NO: 233 was synthesized as appropriate. Basic structures were also prepared and named D-IB1 (s) (SEQ ID NO: 122) and D-TAT (SEQ ID NO: 5), respectively.

  All-D and L fusion peptides were generated by conventional Fmock synthesis and further analyzed by mass spectrometry. They were finally purified by HPLC. To determine the effect of the proline linker, two TAT peptides were made, one with two prolines and the other with no proline. The addition of two prolines did not change the invasion or intracellular localization of the TAT peptide.

(Example 2: Synthesis of all D-retroinverso type IB (s) peptide and its variant)
The peptides of the present invention may be peptides that are all D-amino acids synthesized in the opposite direction (ie, all-D-retroinverso peptides) to prevent proteolysis that occurs in nature. The all-D-retroinverso peptide of the present invention is a peptide having functionality similar to that of the native peptide, and the side groups of the component amino acids are consistent with the natural peptide alignment, but the protease-resistant peptide Peptides that retain the sex backbone are provided.

  The retro-inverso peptides of the present invention can be modified so that the amino acid sequence of the retro-inverso peptide analog is exactly opposite to the amino acid sequence of the selected peptide that functions as a model. It is an analog synthesized using D-amino acids by binding. By way of illustration, when a naturally occurring TAT protein (formed with L-amino acids) has the sequence GRKKRRQRRR (SEQ ID NO: 3), a retro-inverso peptide analog of this peptide (formed with D-amino acids) ) Has the sequence RRRQRRKKRG (SEQ ID NO: 5). Techniques for synthesizing D-amino acid chains to form retro-inverso peptides are known in the art (eg, Jameson et al., Nature, 368, 744-746 (1994); Brady et al. , Nature, 368, 692-693 (1994); see Guichard et al., J. Med. Chem. 39, 2030-2039 (1996)). Specifically, retropeptides were generated by conventional Fmock synthesis and further analyzed by mass spectrometry. They were finally purified by HPLC.

  The problems inherent to natural peptides are degradation by natural proteases and inherent immunogenicity, so that the heterobivalent or heteromultivalent compounds of the present invention include the “retroinverso isomers” of the desired peptides. To be prepared. It protects the peptide from proteolysis that occurs in nature, extends the half-life, and reduces the degree of immune response that helps to actively destroy the peptide, thereby reducing the specific heterobivalent or heteromultivalent The effect of the compound should be enhanced.

Example 3: Long-term biological activity of all-D-retroinverso IB (s) peptides and variants thereof
D-TAT-IB1 (s) (XG-102; dqsrpvqpflnlttpprprrrrrkrkrkgrg when compared to the native L-amino acid analog resulting from protecting the D-TAT-IB (s) peptide from degradation by the native protease ; SEQ ID NO: 233) predicts long-term biological activity of retroinverso-containing peptide heteroconjugates (see chimera sequence above).

  Inhibition of IL-1-induced pancreatic beta cell death by D-TAT-IB1 (s) peptide was analyzed. To TC-3 cells, the designated peptide (1 μmol / L (μM)) was added once and incubated for 30 minutes, and then IL-1 (10 ng / mL) was added.

  The apoptotic cells after 2 days incubation with IL-1 were then counted by using propidium iodide and Hoechst 33342 nuclear staining. A minimum of 1,000 cells was counted for each experiment. The D-TAT-IB1 peptide reduced IL-1-induced apoptosis to the same extent as the L-TAT-IB peptide.

  Long-term inhibition of IL-1P induced cell death by D-TAT-IB1 peptide was also analyzed. Add the specified peptide (1 μmol / L (μM)) once to TC-3 cells and incubate for 30 minutes, then add IL-1 (10 ng / mL), then add cytokines once every two days did. The apoptotic cells after 15 days incubation with IL-1 were then counted by using propidium iodide and Hoechst 33342 nuclear staining. It should be noted that a single addition of TAT-IB1 peptide does not provide long-term protection. A minimum of 1,000 cells was counted for each experiment. As a result, D-TAT-IB1 (s) was able to protect cells over a long period (15 days), but not L-TAT-IB1 (s) peptide.

Example 4: Evaluation of therapeutic activity of D- and L-TAT-IB1 (s) peptides when used according to the present invention
a) Test system:
i) Species / strain: mouse / BALB / c
ii) Supplier: Harlan Israel, Ltd.
iii) Sex: female iv) Total number of animals: n = 150
v) Age: young adult, 7 weeks old at the start of the experiment vi) Body weight: the difference in body weight of the animals at the start of the treatment does not exceed ± 20% of the average body weight vii) Animal health: the health of the animals used in this experiment Status is assessed on arrival and only animals in good health are acclimatized to laboratory conditions (at least 7 days) and used for experiments.
viii) Randomization: Animals were randomly assigned to experimental groups according to a random number table.
ix) Termination: Animals alive at the end of the experiment are euthanized by cervical dislocation.

b) Composition of test groups and dose levels The following table shows the experimental groups included in the experiment.

c) Test procedure Colitis was induced by administering TNBS dissolved in 50% ethanol.

  All animals were then treated intraperitoneally or subcutaneously with doses of XG-102 ranging from 0.1 [mu] g / kg to 1,000 [mu] g / kg as single or repeated daily doses (above).

d) Observations and examinations i) Clinical signs During the experimental period, clinical examinations were carefully performed and recorded. For example, the appearance of skin, hair, eyes, mucous membranes, the occurrence of secretion and excretion (eg, diarrhea), and changes in autonomous activity were observed. In addition to changes in response to walking, posture, and handling, abnormal behavior, tremors, convulsions, sleep, and occurrence of coma were also recorded.

ii) Body weight Individual body weights of animals were measured daily.

iii) Clinical evaluation of colitis Body weight, stool stiffness, and transrectal bleeding were all recorded daily and used as parameters for disease severity scores.

iv) Macroscopic pathology of the colon On the last day of the experiment, the animals were euthanized and the colon was removed for macroscopic pathological evaluation according to the following scores.

e) Results i) Clinical signs No abnormalities were observed during the clinical examination after treatment with XG-102 (SEQ ID NO: 233).

ii) Mortality No death was observed.

iii) Weight TNBS induced significant weight loss on day 1. Administration of XG-102 (SEQ ID NO: 233) prevented weight loss or resolved symptoms and supported healing.

iv) Clinical score The vehicle-treated animals injected with TNBS reached the maximum score on the first day of the experiment and were completely cured only on or after the fifth day of the experiment. Sulfasalazine treatment decreased clinical scores. XG-102 (SEQ ID NO: 233) (single dose or daily dose) administered using any dose, route, or time schedule as defined above is observed with sulfasalazine, a commonly used reference drug The effect more than was done was obtained.

v) Gross pathology score Visual analysis at the end of the experiment revealed that vehicle-treated animals injected with TNBS were damaged by edema and ulcers along the colon. Sulfasalazine was effective in reducing the gross pathological score.

vi) Colon length No effect of disease induction or treatment on colon length was seen.

vii) Colon Weight There was no effect of disease induction or treatment on colon length.

f) Conclusion With the above findings limited to survival data obtained under the above experimental conditions, the exemplary sequence XG-102 according to SEQ ID NO: 233 administered SC or PO promotes disease healing Had activity.

(Example 5: Activity measurement of all-D-retroinverso type IB (s) peptide and its variant in treatment of chronic obstructive pulmonary disease (COPD))
To measure the activity of an exemplary all-D-retroinverso IB (s) peptide (SEQ ID NO: 233) in the treatment of chronic obstructive pulmonary disease (COPD), XG-102 (SEQ ID NO: 233) was used. Used in animal models of bleomycin-induced acute lung inflammation and fibrosis. Protocols for inducing inflammation and fibrosis with bleomycin have already been described in the literature. The purpose of the experiment is
For bronchoalveolar lavage (BAL) and pulmonary neutrophil mobilization in bleomycin-induced inflammation and fibrosis on day 1 after a single dose of bleomycin (10 mg / kg) and on day 10 when fibrosis developed The purpose was to investigate the effect of XG-102 (SEQ ID NO: 233) by the subcutaneous (sc) route.

1) Methods and experimental approaches Two doses of test compound XG-102 (SEQ ID NO: 233) and vehicle control were administered s. c. The mice were analyzed after 1 day and 10 days after dosing. Ten C57BL / 6 mice (8 weeks old) per group were used as model animals. The experimental group included vehicle, 0.001 mg / kg XG-102 (SEQ ID NO: 233) and 0.1 mg / kg XG-102 (SEQ ID NO: 233), treatment was prior to bleomycin administration, then 3 days XG-102 (SEQ ID NO: 233) once subcutaneously. At 24 hours, acute lung inflammation was monitored by BAL lavage, cytodiagnosis, cell count, and lung myeloperoxidase activity. The effect of the compound was compared to the vehicle control. Ten days after a single dose of bleomycin, pulmonary fibrosis was assessed histologically using hematoxylin and eosin staining.

1.1) Bleomycin Administration A bleomycin sulfate-saline solution (10 mg / kg body weight) or physiological saline manufactured by Bell Laboratories (Montrouge, France) by instilling a volume of 40 μL under light ketamine-ketamine anesthesia into the nasal cavity. Water was administered. Bleomycin administration for both bleomycin-induced inflammation and fibrosis included the following: vehicle, 0.001 mg / kg XG-102 (SEQ ID NO: 233) and 0.1 mg / kg XG-102 (SEQ ID NO: 233). ). The route of administration for bleomycin-induced inflammation was the subcutaneous (sc) route, administered as a single dose. The route of administration for bleomycin-induced fibrosis was the subcutaneous (sc) route, administered three times over 10 days.

1.2) Bronchoalveolar lavage fluid (BALF)
After incision of the trachea, a plastic cannula was inserted and the air cavity was washed with 0.3 mL of PBS solution and heated to 37 ° C. The collected sample was divided into two fractions: the first fraction (1 mL corresponding to the first two washes) was used for mediator measurement and the second fraction was used for cell determination. (4 mL). The first fraction was centrifuged (600 g for 10 minutes), the supernatant was fractionated and stored at −80 ° C. until mediator measurement. The cell pellet was then resuspended in 0.4 mL sterile NaCl (0.9%), stored with the second fraction, and used for cell counting.

1.3) Lung homogenate After BAL, all lungs were removed and placed in a microtube (Lysing matrix D, Q Bio Gene, Illkrich, France) with 1 mL of PBS and Fastprep® system (FP120, Q Bio). Gene, Illkrich, France), and then the whole lung tissue extract is then centrifuged, and the extract is centrifuged at −80 ° C. until mediator measurement and collagen assay using Sircol Collagen Assay (France Biochem Division, France) The supernatant was saved.

1.4) Cell number and determination The total cell number in the BAL fluid was determined using a Malassez hemacytometer. After staining with MGG Diff-quick (Dade Behring AG), a differential cell count was performed on cytospin specimens (Cytospin3, Thermo Shandon). Differential cell counts were performed on 200 cells using standard morphological criteria.

1.5) TNF measurement The TNF concentration in BALF was measured using an ELISA assay kit (Mouse DuoSet, R & D system, Minneapolis, USA) according to the manufacturer's instructions. Results are reported in pg / mL.

1.6) MPO measurement MPO concentration was measured when XG-102 was administered. In this experiment, MPO was not significantly induced after bleomycin administration. Furthermore, XG-102 had no effect on MPO concentration in the lung.

1.7) Histology After BAL and pulmonary perfusion, large leaves were fixed with 4% buffered formaldehyde for standard microscopic analysis. 3-m sections were stained with hematoxylin and eosin (H & E).

2. ) Results A) First experiment: Bleomycin (BLM) induced acute lung inflammation Group: Vehicle, XG-102 (SEQ ID NO: 233) 0.001 mg / kg, and XG-102 (SEQ ID NO: 233) 0.1 mg / kg
Route: s. c. Route, single dose

a) Cell recruitment in bronchoalveolar lavage space 0.1 mg / kg XG-102 (SEQ ID NO: 233) significantly reduces neutrophil recruitment and the total number of cells recruited during the inflammatory phase. 0.001 mg / kg XG-102 (SEQ ID NO: 233) has no effect on neutrophil recruitment, or recruitment of other cell types to the bronchoalveolar space (in one exemplary experiment, 1 Five mice per group are used; ^* , p <0.05; ^** , p <0.001).

b) Intrapulmonary cell mobilization using MPO in lung homogenates Myeloperoxidase (MPO) plays an important role in the host defense system. The 140 kDa protein consisting of two 53 kDa heavy chains and two 15 kDa light chains was first discovered in the 1960s. Release of MPO from neutrophil and monocyte granules in response to leukocyte activation converts hydrogen peroxide and chloride ions to hypochlorous acid (HOCl), a powerful oxidant. The MPO serves an important purpose in the defense system, but MPO also plays a role in several inflammatory conditions, for example, various studies have shown that elevated MPO levels are associated with coronary artery disease. Indicated by. In addition, tissue MPO concentration reflects the state of neutrophil activation and provides an indicator of neutrophil tissue infiltration.

  In this experiment, MPO was not significantly induced after bleomycin administration. Therefore, XG-102 (SEQ ID NO: 233) had no effect on MPO concentration in the lung.

c) TNF measurement When TNF concentration was measured, TNF concentration in BALF tended to decrease after XG-102 (SEQ ID NO: 233) administration, but TNF concentration after BLM administration was very low.

d) Conclusions 0.1 mg / kg XG-102 (SEQ ID NO: 233) could be observed to induce a tendency to reduce neutrophil and total cell recruitment to the bronchoalveolar space and reduce TNF concentration. . Furthermore, experiments with histological slides have shown that the accumulation of inflammatory cells in the peribronchial space is reduced. Therefore, it can be concluded that XG-102 (SEQ ID NO: 233) reduces bleomycin-induced inflammation.

  Further experiments were performed in a fibrosis model according to the results obtained.

B) Second experiment: bleomycin (BLM) -induced pulmonary fibrosis Group: vehicle, XG-102 (SEQ ID NO: 233) 0.001 mg / kg, and XG-102 (SEQ ID NO: 233) 0.1 mg / kg
Route: s. c. Route 3 times in 10 days

a) Cell mobilization in the bronchoalveolar lavage space 0.001 mg / kg XG-102 (SEQ ID NO: 233) is a lymphocyte mobilization and total cells mobilized during the inflammatory phase characterized by lymphocyte mobilization in this regard Number was significantly reduced. 0.1 mg / kg XG-102 (SEQ ID NO: 233) had no effect (5 mice per group; ^* , p <0.05; ^** , p <0.001).

a) Histology 3 μm lung sections were stained with hematoxylin and eosin. Ten days after BLM administration, inflammatory cell accumulation, fibrosis area, and loss of lung structure were observed. A decrease in these parameters was observed after administration of the low dose (0.001 mg / kg) of XG-102, but not at the high dose (0.1 mg / kg).

b) Conclusion:
It can be concluded that XG-102 (SEQ ID NO: 233) administered three times at a low dose of 0.001 mg / kg reduces inflammation after bleomycin induction, in particular lymphocyte recruitment was observed at this time. Furthermore, test substances administered three times at low dose attenuate bleomycin-induced fibrosis. It was possible to observe that there were many preserved lung structures and the fibrosis area was not so enlarged.

(Example 6: Activity measurement of all-D-retroinverso type IB (s) peptide and its variant in treatment of Alzheimer's disease)
To measure the activity of an exemplary all-D-retroinverso IB (s) peptide XG-102 (SEQ ID NO: 233) in Alzheimer's disease, in addition to the Morris water maze behavioral test, the amount of plaque is measured APP751-overexpressing hAPP transgenics with London and Swedish mutations using immunohistological studies and ELISA tests that measure the amount of β-amyloid _1-40 and β-amyloid _1-42 in the mouse brain XG-102 (SEQ ID NO: 233) was evaluated in a mouse model.

a) Method i) Introduction Effectiveness of test substance (XG-102, SEQ ID NO: 233) on behavioral, biochemical and histological markers using 5 month (± 2 weeks) old female hAPP Tg mice This experiment was designed to evaluate sex. Therefore, mice were treated once every 2 or 3 weeks for up to 4 months and behavior was assessed in the Morris water maze at the end of the treatment period. At the time of sacrifice, brain, CSF, and blood were collected. The amounts of Aβ40 and Aβ42 in the CSF of four different brain homogenate fractions and Tg mice were measured. The amount of plaque in the cortex and hippocampus of 8 Tg mice per treatment group was quantified.

ii) Animals May (± 2 weeks) old female Tg mice with a background of C57BL / 6xDBA were randomly assigned to treatment groups 1-3 (n = 12). Starting at 5 months of age, animals were administered subcutaneously (sc) once every two or three weeks with vehicle or two different concentrations of XG-102 (SEQ ID NO: 233) for up to 4 months. All animals used in this experiment had dark eyes and were considered to perceive landmarks outside the MWM pool. However, for all animals, including those left in the cage for the experiment, low vision animals that were examined in visual platform training prior to the start of treatment, so-called preliminary tests, had to be excluded. If visual impairment was confirmed in a particular animal, the mouse was excluded from the experiment.

iii) Identification and rearing of animals Mice were individually identified by ear marks. Mice were housed in individual ventilation cages (IVC) on standard rodent bedding supplied by Rettenmeier®. A maximum of 5 mice were housed in each cage. Mice were maintained according to JSW standard operating procedures (SOP GEN011) written according to international standards. Each cage was identified by a colored card indicating the experiment number, gender, animal identification number (IRN), date of birth, and date of screening, and treatment group assignment. During the experiment, the temperature was maintained at about 24 ° C. and the relative humidity was maintained at about 40% to 70%. Animals were housed under a constant light cycle (12 hours light / dark). The animals were able to drink regular tap water freely.

iv) Treatment Forty (40) female hAPP transgenic mice were administered 0.1 mg / kg once every two weeks with 2 different doses of test substance XG-102 (SEQ ID NO: 233) for 4 months. . w. Or 10 mg / kg once every 3 weeks b. w. Treatment (n = 12 / group) or vehicle once every 3 weeks. c. Processed (n = 12).

v) Morris Water Maze (MWM)
Morris water maze (MWM) test was performed in a black circular pool with a diameter of 100 cm. The tap water was filled with a temperature of 22 ± 1 ° C., and the pool was virtually divided into four zones. A transparent platform (8 cm diameter) was placed about 0.5 cm below the water surface. During the entire test period, excluding preliminary tests, the platform was located in the southwest quadrant of the pool. The day before the 4-day training period, the animals had to perform a so-called “preliminary test” (2 trials for 60 seconds) to ensure that each animal had normal vision. Only animals that performed this task were used in the MWM test. In the MWM task, each mouse had to perform 3 trials for 4 consecutive days. One trial lasted for a maximum of 1 minute. During this time, the mice had the opportunity to find a hidden transparent target. If the animal was unable to find a “way” from the water, the investigator guided the mouse to the platform or placed it on the platform. After each trial, the mouse was allowed to rest on the platform for 10-15 seconds. During this time, the mice had an opportunity to look around and confirm their position. In order not to adversely affect the tracking system (Kaminski; PCS, Biomedical Research Systems), the investigation was performed under dim light conditions. A poster with black bold geometric symbols (eg, circles and squares) is affixed to the wall around the pool, and the mouse should use that symbol as a landmark to locate it. I was able to. One swimming group per trial consisted of 5 to 6 mice to ensure a trial interval of about 5 minutes to about 10 minutes. Escape latency (time required to find the platform where the mouse is hidden and escape from the water (seconds)), path (length of the trajectory to reach the target (meters)) and in the target quadrant A computerized tracking system was used to quantify the residence time. A computer was connected to a camera located in the upper center of the pool. The camera detected the signal of a light emitting diode (LED) fixed to a small hairpin with a mouse tail. One hour after the last trial on day 4, the mice had to perform a so-called probe trial. At this time, the platform was removed from the pool and during a one minute probe trial, the experimenter counted the number of times the target had previously passed through. Furthermore, the stay time in this quadrant and the stay time in the other three quadrants were calculated. Through this trial, the mouse was unable to get any clues from the platform no matter what properties it had.

vi) Tissue sampling All surviving mice (n = 28) were sacrificed at the end of the treatment period and after all behavioral tests. Therefore, all mice were sedated by standard inhalation anesthesia (Isofluran, Baxter) as described in SOP MET030. Cerebrospinal fluid (CSF) was obtained by blunt dissection and exposure of the large occipital foramen. Upon exposure, a Pasteur pipette was inserted at a depth of about 0.3 mm to 1 mm in the large occipital foramen. CSF was collected by aspiration and capillary action until the flow stopped completely. Two aliquots of each sample were immediately frozen and stored at −80 ° C. until ready for further analysis using ELISA techniques. After sampling the CSF, each mouse was placed in the dorsal position, the thorax was opened, and a 26 gauge needle attached to a 1 cc syringe was inserted into the right ventricle. The needle was gently aspirated and blood was collected in EDTA and used to obtain plasma. To obtain plasma, rotate each mouse blood sample at 1,750 rpm (700 g) for 10 minutes in a centrifuge (GS-6R Beckman) using a rotor with swing bucket (GH-3.8 Beckman). I let you. Plasma was frozen and stored at −20 ° C. until further analysis. After sampling the blood, the hearts of the transgenic mice were perfused with 0.9% sodium chloride. The brain was quickly removed and the cerebellum was excised. The right hemisphere of all mice was fixed by immersing in freshly made 4% paraformaldehyde / PBS (pH 7.4) for 1 hour at room temperature. Thereafter, the brain was transferred to a 15% sucrose PBS solution and immersed for 24 hours to ensure cryoprotection. The next day, brains were frozen in isopentane and stored at −80 ° C. until used for histological investigation (SOP MET042). The left hemisphere was weighed, frozen in liquid nitrogen and stored at −80 ° C. for biochemical analysis.

Measurements four different brain homogenate fractions of each Tg mouse vii) A [beta] _1-40 and A [beta] _1-42, and A [beta] _1-40 and A [beta] _1-42 levels in CSF samples were assessed using an ELISA technique . High sensitivity Aβ _1-40 and Aβ _1-42 ELISA test kits were purchased from The Genetics Company ™, Switzerland (SOP MET058). CSF was prepared as described above. Frozen hemispheres of brain homogenate were homogenized in TRIS buffered saline (TBS) -buffer (5 mL) containing a protease inhibitor cocktail. 1.25 mL of this initial brain TBS homogenate was stored at −80 ° C., and 1.25 mL was further examined. The remaining brain homogenate (2.5 mL) was centrifuged, and the resulting supernatant (= TBS fraction) was dispensed and stored at −20 ° C. until ELISA measurement. The pellet was suspended in Triton X-100 (2.5 mL), centrifuged, and the supernatant (= Triton X-100 fraction) was dispensed and stored at −20 ° C. These steps were repeated using SDS (2.5 mL). The pellet of the SDS fraction was suspended in 70% formic acid (0.5 mL) and then centrifuged. The obtained supernatant was neutralized with 1 mol / L (M) TRIS (9.5 mL), dispensed, and stored at −20 ° C. (= FA fraction). Samples of 4 brain homogenate fractions (TBS, Triton X-100, SDS, and FA) were used for Aβ _1-40 and Aβ _1-42 measurements using ELISA technology. The ELISA test kit was purchased from The Genetics Company ™, Switzerland (SOP MET062). It could be inferred that TBS and Triton X-100 solubilized the monomer-oligomer structure. Polymer-like prefibrils and water-insoluble fibers could be dissolved in SDS and FA. In this regard, examining all four fractions gives insight into the polymerization state of A.

vii) Evaluation of brain morphology All Tg animal brain tissues investigated were manipulated in exactly the same way to avoid bias due to variability in this procedure. Twenty frozen sections (Leica CM 3050S) per layer (5 layers in total) each having a thickness of 10 μm were cut in a sagittal manner from half of the brains of 24 Tg mice (8 mice in each group). Sheets (one from each layer) were processed and evaluated to quantify the amount of plaque. The five sagittal layers are shown in FIGS. 104-105, 107-108, 111-112, 115-116, and 118-118 of the morphological atlas "The Mouse Brain" (2nd edition) by Paxinos and Franklin. Corresponded to 119. The first layer was identified by the requirement to include the entire hippocampus with regions CA1, CA2, CA3, GD1b, and GDmb. Immunoreactivity was quantitatively assessed in the hippocampus and cortex using monoclonal human Aβ-specific antibody 6E10 (Signet) and thioflavin S staining. The remaining brain hemisphere or tissue that was not used was stored in JSW CNS until the end of the project.

b) Evaluation i) Behavior In the Morris water maze trial, the length of the swimming distance, the escape latency, the swimming speed, the number of times the platform passed through the previous position in the probe trial, and the staying time in each quadrant of the pool Measured for each Tg animal using special computer software.

ii) Biochemical evaluation All Tg mouse CSF samples and brain specimen samples were analyzed using commercially available Aβ _1-40 and Aβ _1-42 ELISA. Appropriate standard measurements were performed simultaneously. Brain specimen samples were analyzed in duplicate. Since there was only a small amount, the CSF sample could only be measured once.

iii) Histology il) Measurement of amyloid deposition and plaque mass The following evaluation procedure was used for 6E10 immunohistochemistry:
aa) Contrast images to visualize segment boundaries without applying contrast to the images.
bb) Interactive drawing of the cortical outline and measuring the cortical area (= area of the area).
cc) Interactive drawing of the area of interest (AOI), where a stained object is detected that exceeds a certain intensity and is greater than 8 μm ² based on a threshold level (same for each image) .
dd) Measure the number of objects after smooth contrast to increase the area of each object, the sum of the stained areas in the AOI, and the signal / noise ratio (same for each image).
ee) Repeat aa) to dd) for the hippocampus.
ff) Average plaque size (= “total plaque area / number of plaques”), relative plaque number and area (= “number of plaques / region area” and “total plaque area / region area × 100”) calculate.
gg) Automatically export data including parameters “image title, area area, number of plaques, total area of plaques, relative number of plaques, relative plaque area, and average plaque size” to an Excel spreadsheet. The image quality and exclusion criteria were recorded, respectively, using the findings field. Exclusion criteria were missing sections, wrinkled, large scratches, or staining inconsistencies (eg, due to swelling that could hinder adequate reaction of the blocking reagent).
hh) Close the image without saving (to save the raw data as is).

c) Results i) Overall observation A total of 40 female hAPP Tg mice were used in the experiment. For unknown reasons, 12 of these mice died before the end of the treatment period.

ii) Results of behavior Spatial learning in MWM was not widely affected by XG-102 (SEQ ID NO: 233) processing. The 0.1 mg / kg-treated mice showed a tendency that the learning results decreased during the first to fourth days. Two-way ANOVA on average performance from day 1 to day 4, not only significantly significant learning (p <0.001) in all groups, but also significant effect of factor treatment (p = 0.045) It was revealed. However, no significant difference was reached after correction by Bonferroni method.

iii) Biochemical results aa) Aβ amount in brain homogenate fraction Treatment with test compound XG-102 (SEQ ID NO: 233) did not affect the amount of Aβ _1-40 in brain homogenate. A group difference of Aβ _1-40 amount was observed only with Triton X-100. These animals treated with the low dose test compound XG-102 (SEQ ID NO: 233) (0.1 mg / kg) were divided into vehicle group (p <0.05) and high dose group (P <0.01). A significant decrease was shown.

bb) Aβ amount of CSF After treatment with test substance XG-102 (SEQ ID NO: 233), the amounts of Aβ _1-40 and Aβ _1-42 were significantly decreased in CSF compared to the vehicle group. The p-values for both Aβ _1-40 and Aβ _1-42 were p <0.01 at the high dose (10 mg / kg) of XG-102 (SEQ ID NO: 233) and p <0.05 at the low dose. It was.

iv) Brain tissue and immunohistological results aa) Amyloid deposits and plaque mass The plaque mass was quantified by two different methods. On the one hand, IHC staining using 6E10 was mainly performed on AA1-17 of human amyloid peptide, and on the other hand, thioflavin S staining for labeling the core of β-sheet structure and mature senile plaques was performed. First of all, the area of the measured area (cortex and hippocampus) is very constant in all groups, which can rule out problems in amputation and IHC procedures, and the treatment induced some atrophy. (This method detects more than 5% change). Quantification of 6E10 and thioflavin S selectively reduced β-sheet structure in the plaque center after XG-102 (SEQ ID NO: 233) treatment, but human amyloid was unaffected or slightly increased by treatment It became clear. Specifically, the amount of cortical 6E10 IR plaques was increased over vehicle in mice treated with 10 mg / kg XG-102 (SEQ ID NO: 233), but reached a significant level for the number of hippocampal plaques. It was. In contrast to 6E10 IHC, XG-102 (SEQ ID NO: 233) treatment led to a negative dose-dependent decrease in the number of hippocampal thioflavin S positive plaques and the proportion of areas (number of plaques: XG-102). (SEQ ID NO: 233) p <0.05 at 10 mg / kg, p <0.01 at 0.1 mg / kg). In addition, treatment with 0.1 mg / kg XG-102 (SEQ ID NO: 233) reduced mean plaque size, but this effect was significant in ANOVA (unpaired, two-tailed T-test: p = 0.074). I did not reach the right level. These effects are not seen for cortical plaques, probably because the pathology of plaques in the hippocampus develops later than the cortex. If treatment is started at 5 months of age, the time of hippocampal plaque deposition is accurately captured, while cortical plaques begin to appear at the magnification used to quantify at 3 months of age. The ratio of 6E10 to thioflavin S stained plaques increases quantitatively, and the β-sheet plaque core size begins to decrease, as seen in the double-labeled sections. In summary, these data suggest that XG-102 (SEQ ID NO: 233) treatment acts on β-sheet formation in the early phase of plaque deposition and β-sheet formation in the plaque core, respectively. .

d) Summary of effects and conclusions • Spatial navigation measured in the Morris water maze was not widely affected by the treatment. The 0.1 mg / kg XG-102 (SEQ ID NO: 233) treatment slightly reduced the learning performance between the start date and the last day of training.
_-The amount of Aβ _1-40 and Aβ _1-42 in the brain was stable except that it decreased in the Triton X-100 fraction of the 0.1 mg / kg XG-102 (SEQ ID NO: 233) group.
• A decrease in the amount of Aβ in CSF was detectable for both doses and fragments.
• XG-102 (SEQ ID NO: 233) treatment led to an increasing trend in human amyloid β in the high dose group at 6E10 quantification, consistent with the data obtained in the Aβ ELISA.
The amount of hippocampal β-sheet detected by thioflavin S staining decreased in a dose-dependent manner after treatment with XG-102 (SEQ ID NO: 233) (low dose 0.1 mg / kg XG-102 (SEQ ID NO: 233)) In contrast, the amount of cortical plaques remained unchanged. It is suggested that the age-dependent onset of plaque deposition in the hippocampus at the start of treatment implies an early effect on β-sheet formation in the early phase of plaque deposition.

(Example 7: Activity measurement of all-D-retroinverso type IB (s) peptide and its variants in the treatment of type 2 diabetes)
The purpose is to measure the activity of IB (s) peptide, all-D-retroinverso IB (s) peptide and its variants in the treatment of type 2 diabetes, specifically 1 every 3 days. The purpose was to determine the effect of chronic treatment with XG-102 (SEQ ID NO: 233) in a db / db mouse model of type 2 diabetes by assessing fasting (28 days) fasting blood glucose concentration.

a) Materials and Methods i) Animals A total of 20 male db / db mice (8 weeks old) were purchased from Charles River (Germany). Upon arrival, animals are housed (n = 6-7 / group), unless otherwise specified, with regular rodent diet (Altromin standard # 1324 chow; C. Petersen, Ringsted, Denmark) and water ad libitum It was.
Mice were housed in a temperature and humidity controlled room under a 12:12 L / D cycle (lights up at 4:00 and turns off at 16:00).

ii) Groups and randomization On day 4, mice were randomized according to blood glucose concentration (fasting; blood glucose measured using Biosen Sline analyzer (EKF diagnostic, Germany)) and the following drugs Assigned to one of the treatment groups (n = 6):
1) Vehicle control, S.I. C. (Physiological saline)
2) XG-102 (SEQ ID NO: 233); 1 mg / kg; s. c.
3) XG-102 (SEQ ID NO: 233); 10 mg / kg; s. c. .
All the above doses were calculated for the free base. The purity of the drug was 95.28% and the peptide content was 78.0%. All compounds were administered subcutaneously (sc) in a volume of 3 mL / kg. The formulation procedure for vehicle control and XG-102 (SEQ ID NO: 233) was as follows.

First, XG-102 (SEQ ID NO: 233) was dissolved in a vehicle. The formulations (concentrations of 0.33 mg / mL and 3.3 mg / mL, corresponding to doses of 1 mg / kg and 10 mg / kg, respectively) were prepared according to the procedure detailed below. Concentrations were calculated and expressed taking into account the purity and peptide content of the test items (multiplication count was 1.346).
-Preparation of stock solution: Lyophilized test compound XG-102 (SEQ ID NO: 233) was thawed for a minimum of 1 hour to prepare a vehicle stock solution of 1 mmol / L (mM) (corresponding to 3.823 mg / mL). Aliquots were prepared for each treatment day and stored at about -80 ° C. On each treatment day, the stock solution is diluted to the required concentration.
-Stock solution storage: about -80 ° C;
-Storage of diluted preparation: 24 hours at maximum room temperature.

  Prior to solubilization, the powder was stored at -20 ° C. The stock solution is stable at about −80 ° C. for 3 months, and the diluted formulation for administration to animals is stable for 24 hours at room temperature. Diluted material that was not used could be stored for up to 7 days when stored at 4-8 ° C.

c) Experimental procedure After acclimatization for 8 days, mice were treated once a day at AM 8:00 for 21 days by SC administration 8 hours before extinguishing at PM 4:00 according to the outline group. Then, on day 21 of the experiment, administration of the highest concentration of XG-102 (SEQ ID NO: 233 (10 mg / kg)) was stopped, but one day of vehicle control and XG-102 (SEQ ID NO: 233 (1 mg / kg)). The single administration was continued until the 28th day of the experiment. From the 28th day to the 111th day, the vehicle treated with vehicle and XG-102 (SEQ ID NO: 233 (10 mg / kg)) in the washout period was observed, but XG-102 (SEQ ID NO: 233) was observed. Mice treated with (1 mg / kg)) were terminated 28 days after treatment.

i) Blood glucose Blood glucose was administered to animals fasted for 7 hours by collecting 10 μL of blood sample from the tail vein into a hematocrit tube and then analyzing with Biosen s-line analyzer (EKF-diagnostic; Germany). Blood glucose was measured 6 hours later.

ii) Metabolic cages Group 1 + 3: In addition to 24-hour food and water intake, mice were placed in metabolic cages to record 24-hour urine and fecal excretion. Mice were stratified into two subteams of n = 6-7, and then metabolic characterization was performed from experiment days 71-72.

iii) Adipokine Panels Group 1 + 3: Blood was collected from the tail vein 3 times (Experiments 57, 66, and 108) using EDTA-coated hematocrit tubes (100 μL). After centrifuging the blood, plasma was collected and stored at −20 ° C. until measurement. The following panel of adipokines / cytokines was then measured using the Luminex line 7-plex: leptin, resistin, MCP-1, PAI-1, TNFα, insulin, and interleukin-6 (IL-6).

iv) Termination Group 1 + 3 (day 111): The following organs were excised and weighed: subcutaneous fat in the groin, epididymal fat, retroperitoneal fat, brain, liver, kidney, spleen and heart. All organ samples were fixed with 4% PFA for future histopathological evaluation. In addition, the pancreas (generally) was sampled to allow steric analysis and immunohistochemical analysis, and the eye was sampled for later analysis of retinopathy. Group 2 (Day 28): No tissue or plasma was collected.

c) Results i) Overall observation During the acute dosing period, the animals showed normal levels of arousal and activity, and no signs of sedation were seen in the drug treated animals. Diet and water intake of vehicle-treated animals was within normal limits. However, approximately 2 weeks after administration, nodular fibrosis was observed in the subcutaneous tissue in response to high doses of XG-102 (SEQ ID NO: 233) compound, which progressed to open wounds in all mice in group C did. In group B, mild nodular fibrosis was observed. As a result, injection sites were used alternately. After completion of the animal administration, the animal was cured and the nodular fibrosis gradually disappeared. There was no clinical effect in vehicle-treated animals.

ii) Blood glucose In groups A and C, fasting blood glucose (absolute and relative concentrations) was measured once every 3 days until day 68, and measured periodically until the end of day 111. Fasting blood glucose levels of diabetic db / db mice treated with XG-102 (SEQ ID NO: 233) (10 mg / kg) are clearly and significantly (p <0.001) lower than vehicle control Was observed. Fasting blood glucose concentrations in mice treated with XG-102 (SEQ ID NO: 233) (10 mg / kg) reached a low plateau of approximately 5 mmol / L (mM). This effect became apparent 14 days after dosing and persisted during the experimental period, ie during the entire washout period from day 21 to day 11. In contrast, during 28 days of administration, no effect was seen with the low dose of XG-102 (SEQ ID NO: 233) (1 mg / kg).

iii) Body weight It was observed that mice treated with XG-102 (SEQ ID NO: 233) (10 mg / kg) clearly and significantly (p <0.001) suppressed body weight gain compared to vehicle control. . This effect was evident on day 28 of administration and persisted until the end of day 111. In contrast, there was no effect on body weight of low dose XG-102 (SEQ ID NO: 233) (1 mg / kg) during 28 days of administration.

iv) Metabolic cages Effect of vehicle or XG-102 (SEQ ID NO: 233) on 24-hour food and water intake and urine and fecal excretion as measured in metabolic cages on day 68 of experiment (weight (g) Was normalized). Although there was a trend towards decreased food intake and urinary excretion, no significant effect of XG-102 (SEQ ID NO: 233) on any of the measured parameters was seen compared to the vehicle control.

v) Adipokine Vehicle or XG- for plasma concentrations of insulin, MCP-1, IL-6, tPAI-1, TNF, and resistin as measured on days 57, 77, and 108 The effect of 102 (SEQ ID NO: 233) (10 mg / kg) was analyzed. On days 77 and 108, against any of the parameters measured when compared to vehicle control, except that plasma resistin concentrations were significantly higher in animals treated with XG-102 (SEQ ID NO: 233) No significant effect of XG-102 (SEQ ID NO: 233) was seen.

vi) Termination tissue weight The effect of vehicle or XG-102 (SEQ ID NO: 233) (10 mg / kg) on the tissue weight of epididymis, subcutaneous groin, and retroperitoneal fat pad was analyzed. Compared to vehicle control, mice treated with XG-102 showed a significant decrease in epididymal (p <0.05) and retroperitoneal (p <0.01) fat mass. The effect of vehicle or XG-102 (SEQ ID NO: 233) (10 mg / kg) on brain, spleen, and heart tissue weights was analyzed. There was no significant effect of XG-102 (SEQ ID NO: 233) (10 mg / kg) on these parameters compared to the vehicle control. Finally, the effect of vehicle or XG-102 (SEQ ID NO: 233) (10 mg / kg) on kidney and liver tissue weights was analyzed. Compared to vehicle control, mice treated with XG-102 (SEQ ID NO: 233) showed a significant decrease in kidney (p <0.05) and liver (p <0.01) mass.

  To summarize the results, administration of XG-102 (SEQ ID NO: 233) (10 mg / kg) appears to significantly reduce blood glucose levels, and XG-102 (SEQ ID NO: 233) is diabetic and hyperblooded. This suggests a promising new tool for treating glucose concentrations.

8). Measurement of peptide uptake into cells (internalization) and peptide internalization into cells In this experiment, the in vitro internalization (uptake) ability of FITC-labeled TAT-derived transporter construct was determined in the cell line HL-60 (leukemia). Evaluation was performed using a fluorescent plate reader.

8.1 Test Samples Used in Experiments The constructs used in this experiment consisted of four different TAT-derived transporter constructs (L-TAT, r3-TAT (also called r3-L-Tat), r3-TATi (r3-L- Also called TATi) and D-TAT), each prepared as described above. These constructs have a length of 9 amino acids but differ in the D- / L-pattern. In addition, a construct DAK containing no transporter sequence was used as a control. The construct was N-terminal protected with β-alanine (βA) and labeled with FITC.
FITC-βA-L-TAT FITC-βA-RKKRRQRRR
FITC-βA-D-TAT FITC-βA-rrrrqrrkkr
FITC-βA-r3-L-TAT FITC-βA-rKKRrQRRr
FITC-βA-r3-L-TATi FITC-βA-rRRQrRKKKr
FITC-βA-DAK (control) FITC-βA-DAK (no transporter sequence)

  The construct was prepared, purified as described above, stored as a 10 mmol / L (mM) sterile aqueous solution, and used as a purified product without any further processing.

8.2 Further transporter constructs TAT (s2-1) -TAT (s2-96)
For the transporter construct of the present invention, further transporter constructs TAT (s2-1) -TAT (s2-96) were widely prepared as described above. Therefore, the following sequences and protecting groups were used during synthesis (bonded to the resin):
TATs2-1: D-Arg (Pmc) -Ala-Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) ) -Resin TATs2-2: D-Arg (Pmc) -Lys (Boc) -Ala-Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D- Arg (Pmc) -resin TATs2-3: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Ala-D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-4: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) Ala-Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-5: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg ( Pmc) -Gln (Trt) -Ala-Arg (Pmc) -D-Arg (Pmc) -resin TATs2-6: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D -Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Ala-D-Arg (Pmc) -resin TATs2-7: D-Arg (Pmc) -Asp (OBut) -Lys (Boc) -Arg (Pmc) ) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-8: D-Arg ( mc) -Lys (Boc) -Asp (OBut) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2- 9: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Asp (OBut) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg ( Pmc) -resin TATs2-10: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Asp (OBut) -Arg (Pmc) -Arg (Pmc) ) -D-Arg (Pmc) -resin TATs2-11: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Ar (Pmc) -Gln (Trt) -Asp (OBut) -Arg (Pmc) -D-Arg (Pmc) -TATs2-resin TATs2-12: D-Arg (Pmc) -Lys (Boc) -Lys (Boc)- Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Asp (OBut) -D-Arg (Pmc) -TATs2-resin TATs2-13: D-Arg (Pmc) -Glu ( OBut) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-14: D-Arg (Pmc) -Lys (Boc) -Glu (OBut) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc ) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-15: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Glu (OBut) -D-Arg (Pmc) -Gln ( Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-16: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Glu (OBut) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-17: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg ( Pmc) -D-Arg (Pmc) -Gln (Trt) -Glu (OBut) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-18: D- Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Glu (OBut) -D-Arg (Pmc) -resin TATs2-19: D-Arg (Pmc) -Phe-Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) ) -Resin TATs2-20: D-Arg (Pmc) -Lys (Boc) -Phe-Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D- Arg (Pmc) -resin TATs2-21: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Phe-D-Arg (Pmc) -Gln (T t) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-22: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Phe-Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-23: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc)- D-Arg (Pmc) -Gln (Trt) -Phe-Arg (Pmc) -D-Arg (Pmc) -resin TATs2-24: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg ( Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Phe-D-Arg (Pmc) -resin TATs2-25: D-Arg (Pmc -Arg (Pmc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-26: D-Arg (Pmc) -Lys (Boc) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -Resin TATs2-27: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Lys (Boc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc)- D-Arg (Pmc) -resin TATs2-28: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc -Arg (Pmc) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-29: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc)- D-Arg (Pmc) -Gln (Trt) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-30: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Lys (Boc) -D-Arg (Pmc) -resin TATs2-31: D-Arg (Pmc) -His (Trt ) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -Resin TATs 2-32: D-Arg (Pmc) -Lys (Boc) -His (Trt) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc)- D-Arg (Pmc) -resin TATs2-33: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -His (Trt) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-34: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) His (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-35: D-Arg (Pmc) -Lys (Boc) -Lys (Boc ) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -His (Trt) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-36: D-Arg (Pmc) -Lys ( Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -His (Trt) -D-Arg (Pmc) -resin TATs2-37: D-Arg (Pmc) -Ile-Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-38: D-Arg (Pmc) -Lys (Boc) -Ile-Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) D-Arg (Pmc) -resin TATs2-39: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Ile-D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg ( Pmc) -D-Arg (Pmc) -resin TATs2-40: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Ile-Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-41: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) ) -Ile-Arg (Pmc) -D-Arg (Pmc) -resin TATs2-42: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -A g (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Ile-D-Arg (Pmc) -resin TATs2-43: D-Arg (Pmc) -Leu-Lys (Boc)- Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-44: D-Arg (Pmc) -Lys (Boc) -Leu-Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-45: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Leu-D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -tree TATs2-46: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Leu-Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) ) -Resin TATs2-47: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Leu-Arg (Pmc) -D- Arg (Pmc) -resin TATs2-48: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Leu -D-Arg (Pmc) -resin TATs2-49: D-Arg (Pmc) -Met-Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-50: D-Arg (Pmc) -Lys (Boc) -Met-Arg (Pmc) -D-Arg ( Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-51: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Met-D -Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-52: D-Arg (Pmc) -Lys (Boc) -Lys (Boc)- Arg (Pmc) -D-Arg (Pmc) -Met-Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-53: DA g (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Met-Arg (Pmc) -D-Arg (Pmc) -resin TATs2-54 : D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Met-D-Arg (Pmc) -resin TATs2-55: D-Arg (Pmc) -Asn (Trt) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D- Arg (Pmc) -resin TATs2-56: D-Arg (Pmc) -Lys (Boc) -Asn (Trt) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-57: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Asn (Trt) -D- Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-58: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Asn (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-59: D-Arg (Pmc) -Lys (Boc)- Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Asn (Trt) -Arg (Pmc) -D-Arg (Pmc) -resin T ATs2-60: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Asn (Trt) -D- Arg, (Pmc) -resin TATs2-61: D-Arg (Pmc) -Gln (Trt) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc)- Arg (Pmc) -D-Arg (Pmc) -resin TATs2-62: D-Arg (Pmc) -Lys (Boc) -Gln (Trt) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-63: D-Arg (Pmc) -Lys (Boc) -Lys (Boc)- ln (Trt) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-64: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) Lys (Boc) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-65: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Gln (Trt) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-66: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -G n (Trt) -D-Arg (Pmc) -resin TATs2-67: D-Arg (Pmc) -Ser (But) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-68: D-Arg (Pmc) -Lys (Boc) -Ser (But) -Arg (Pmc) -D-Arg (Pmc) ) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-69: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Ser (But) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-70: D-Arg (Pm ) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) Ser (But) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-71: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Ser (But) -Arg (Pmc) -D-Arg (Pmc) -Resin TATs2-72: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Ser (But)- D-Arg (Pmc) -resin TATs2-73: D-Arg (Pmc) -Thr (But) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-74: D-Arg (Pmc) -Lys (Boc) -Thr (But) -Arg (Pmc)- D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-75: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Thr (But) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-76: D-Arg (Pmc) -Lys (Boc ) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) Thr (But) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc)- Fat TATs2-77: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Thr (But) -Arg (Pmc) -D -Arg (Pmc) -resin TATs2-78: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc)- Thr (But) -D-Arg (Pmc) -resin TATs2-79: D-Arg (Pmc) -Val-Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg ( Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-80: D-Arg (Pmc) -Lys (Boc) -Val-Arg (Pmc) D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-81: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Val-D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-82: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Val-Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-83: D-Arg (Pmc) -Lys (Boc ) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Val-Arg (Pmc) -D-Arg (Pmc) -resin T Ts2-84: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Val-D-Arg (Pmc) ) -Resin TATs2-85: D-Arg (Pmc) -Trp (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-86: D-Arg (Pmc) -Lys (Boc) -Trp (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc ) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-87: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Trp (Bo ) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-88: D-Arg (Pmc) -Lys (Boc) -Lys ( Boc) -Arg (Pmc) -D-Arg (Pmc) Trp (Boc) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-89: D-Arg (Pmc) -Lys ( Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Trp (Boc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-90: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Trp (Boc) -D-Arg (Pmc) -resin TATs2-91: D-Arg (Pmc) -Tyr (But) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc ) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-92: D-Arg (Pmc) -Lys (Boc) -Tyr (But) -Arg (Pmc) -D-Arg (Pmc) -Gln ( Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-93: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Tyr (But) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-94: D-Arg (Pmc) -Lys ( Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) Tyr (But) -Arg (Pmc) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2-95: D-Arg ( Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Tyr (But) -Arg (Pmc) -D-Arg (Pmc) -resin TATs2- 96: D-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Arg (Pmc) -D-Arg (Pmc) -Gln (Trt) -Arg (Pmc) -Tyr (But) -D-Arg ( Pmc) -resin

8.3 Materials and methods for uptake experiments a) Cell lines:
The cell line used for this experiment was HL-60 (reference number CCL-240, ATCC, lot number 116523).

b) Culture medium and plates 10% FBS (reference number A64906-0098, PAA, lot number A15-151): complement removal for 30 minutes at 56 ° C. on April 4, 2008 1 mmol / L (mM) sodium pyruvate (Reference number S8636, Sigma, lot number 56K2386)
Penicillin (100 units / mL) / streptomycin (100 μg / mL) (reference number P4333, Sigma, lot number 106K2321)
Was supplemented on May 5, 2008 with RPMI (reference number 21875-091, Invitrogen, lot number 8296) or DMEM (reference number 41965, Invitrogen, lot number 13481)
PBS 10X (reference number 70011, Invitrogen, lot number 8277): diluted 1 × with sterile water Trypsin-0.05% EDTA (reference number L-11660, PAA, lot number L6607-1194)
6-well culture plate (reference number 140675, Nunc, lot number 102613)
24-well culture plate (reference number 142475, Nunc, lot number 095849)
96-well culture plate (reference number 167008, Nunc, lot number 083310)
96-well protein dosing plate (reference number 82.1581, Sarstedt)
96-well fluorescence plate (reference number 6005279, Perkin Elmer)

Solution Poly-D-lysine coating solution (Sigma P9011 lot number 095K5104): Finally diluted to 25 μg / mL with 1 × PBS Acid wash buffer: 0.2 mol / L (M) glycine, 0.15 mol / L (M) NaCl (pH 3.0)
Ripa lysis _{buffer: 10mmol / L (mM) NaH} 2 PO 4 (pH7.2) of, 150 mmol / L of NaCl, 1% of (mM) TritonX-100,1mmol / L (mM) EDTA (pH8.0) , 200 μmol / L (μM) Na ₃ VO ₂ , 0.1% SDS, 1 × protease inhibitor cocktail (reference number 11873580001, Roche, lot number 13732700)

d) Microscope and fluorescence plate reader Cells were observed and counted using an inverted microscope (Axiovert 40 CFL; Zeiss; 20X).
Fluorescence was read with a Fusion Alpha Plate reader (Perkin Elmer).

e) Method The internalization of the FITC-labeled peptide was examined in floating cells. Cells were plated on poly-DL-lysine coated dishes at a concentration of 1 × 10 ⁶ cells / mL. The plates were then incubated for 24 hours at 37 ° C., 5% CO ₂ , and 100% relative humidity before adding known concentrations of peptides. After the peptide was added, the cells were incubated for 30 minutes, 1 hour, 6 hours, or 24 hours at 37 ° C., 5% CO ₂ and 100% relative humidity. The cells are then washed twice with acidic buffer (0.2 mol / L (M) glycine, 0.15 mol / L (M) NaCl, pH 3.0) to remove the peptide adsorbed on the cell surface. Washed (see Kameyama et al., (2007), Biopolymers, 88, 98-107). Peptides containing a large amount of basic amino acids strongly adsorb to the cell surface. Due to this, there are many cases where more internalized peptides are presumed than actual, so an acidic buffer was used. Therefore, cells were washed with an acidic buffer to remove peptides adsorbed on the cell surface. An acid wash in the measurement of intracellular uptake of the Fab / cell permeable peptide conjugate was performed and then washed twice with PBS. Cells were disrupted by adding RIPA lysis buffer. The relative amount of peptide internalized by fluorescence was then determined after reducing the background and normalizing the protein content.
The process is as follows:
1. Cell culture 2. Acid washing and cell extraction Analysis of peptide internalization using a fluorescence plate reader.

f) Cell culture and peptide treatment (1) A 6-well culture plate was coated with 3 mL poly-D-Lys (Sigma P9011; 25 μg / mL in PBS), a 24-well plate was coated with 600 μL, and a 96-well plate was 125 μL And incubated at 37 ° C., 5% CO ₂ and 100% relative humidity for 4 hours.
(2) After 4 hours, each 6-well plate, 24-well plate, or 96-well plate was washed twice with 3.5 mL, 700 μL, or 150 μL PBS.
(3) Cells were plated in 2.4 mL medium (RPMI) in the dish at a plating density of 1,000,000 cells / mL (floating cells). After inoculation, the plates were incubated for 24 hours at 37 ° C., 5% CO ₂ and 100% relative humidity before adding the peptide. Adherent cells were considered to be 90% -95% density on the day of treatment and were plated in DMEM.
(4) Cells were treated with a desired concentration of FITC-labeled peptide (stock solution at a concentration of 10 mmol / L (mM) in water).
(5) After peptide addition, the cells were incubated at 37 ° C., 5% CO ₂ , and relative humidity 100% for 0 to 24 hours (eg, 30 minutes, 1 hour, 6 hours, or 24 hours).

Acid washing and cell extraction:
(6) The extract was cooled on ice.
Suspended cells (ie, cells not attached to the dish well):
• Cells were transferred to 15 mL falcon. The dish was washed with 1 mL PBS for maximum cell recovery.
• Cells were collected for 2 minutes at a maximum of 2,400 rpm.
• Cells were suspended in 1 mL cold PBS.
• Cells were transferred to coated “Eppendorf tubes” (coated with 1 mL poly-D-Lys for 4 hours and washed twice with 1 mL PBS).
• Washed 3 times with 1 mL cold acidic wash buffer and centrifuged for 2 minutes at a maximum of 2,400 rpm. Pay attention to the diffusion of cells in the Eppendorf.
-Washed twice with 1 mL cold PBS to neutralize.
• Added 50 μL of lysed RIPA buffer.
Incubated on ice for 30 minutes to 1 hour with stirring.
Adherent cells:
Washed 3 times with 3 mL, 1 mL, or 200 μL of cold acidic wash buffer (for 6-well plate, 24-well plate, or 96-well plate, respectively). Watch out for cells that detach from the dish.
• Neutralized by washing twice with 1 mL cold PBS (for 6 well plates, 24 well plates, or 96 well plates, respectively).
• Added 50 μL of lysed RIPA buffer.
Incubated on ice for 30 minutes to 1 hour with stirring.
• Fragment cells with cold scrapper. 24-well plates and 96-well plates were directly centrifuged at 4,000 rpm for 15 minutes at 4 ° C. to remove cell debris. The supernatant (100 mL or 50 mL for a 24 well plate or 96 well plate, respectively) was then transferred directly to a dark 96 well plate. Plates were read with a fluorescence plate reader (Fusion Alpha, Perkin Elmer).
Transfer the lysate to a coated “Eppendorf tube” (coated with 1 mL poly-D-Lys for 4 hours and washed twice with 1 mL PBS).
-The lysed cells were then centrifuged at 10,000g for 30 minutes at 4 ° C to remove cell debris.
The supernatant was removed and stored at −80 ° C. in a coated “Eppendorf tube” (coated with 1 mL poly-D-Lys for 4 hours and washed twice with 1 mL PBS).

Analysis of peptide internalization using a fluorescence plate reader:
(7) The content of each protein extract was determined by standard BCA assay (Kit N23225, Pierce) according to the manufacturer's instructions.
(8) Read 10 μL of each sample with a fluorescence plate reader (Fusion Alpha, Perkin Elmer), reduce background, normalize by protein concentration, and determine the relative fluorescence of each sample.

8.4 Internalization Experiment and Analysis Using the materials and methods described above, time-dependent internalization (incorporation) of the FITC-labeled TAT-derived transporter construct into cells of the HL-60 cell line was performed.

  Briefly, HL-60 cells were incubated with 10 μmol / L (μM) TAT derivative transporter for 30 minutes, 1 hour, 6 hours, or 24 hours. The cells were then washed twice with acidic buffer (0.2 mol / L (M) glycine, 0.15 mol / L (M) NaCl, pH 3.0) and twice with PBS. Cells were disrupted by adding RIPA lysis buffer. Then, after reading the fluorescence intensity of each extract (Fusion Alpha plate reader; PerkinElmer), the relative amount of internalized peptide was measured by reducing the background and normalizing the protein content. The r3-L-TAT transporter construct showed the same internalization ability as the D-TAT transporter construct. The r3-L-TATi transporter construct that internalizes in a time-dependent manner like both of the transporters described above appears to be preferred, albeit with low efficiency, while L-TAT accumulates over a 24 hour period. I found out that I would not.

  Furthermore, confocal microscopy was performed using cells treated with the fluorescently labeled TAT-derived transporter construct as described above. Dissociated primary cortical neurons from P2 Sprague Dawley rats were cultured in neural basal medium for 12 days and then exposed to 500 nmol / L (nM) FITC-labeled TAT derivative transporter for 24 hours. Cells were washed 5 times with PBS on ice and then encapsulated in fluorsave inclusion medium without prior fixation. Images were acquired using an LSM510 metaconfocal microscope (Zeiss). Images were processed using LSM510 software and mounted using Adobe photoshop. Labeling with 500 nmol / L (nM) FITC-transporter (A: green) was visualized with a confocal microscope. Nuclei were stained with Hoechst (B: blue). In addition to r3-L-TAT, D-TAT and r3-L-TATi transporter constructs were also internalized into the cytoplasm of unstressed neurons (C: merged panel). However, after 24 hours of incubation, the L-TAT transporter was no longer present.

8.5 Further internalization experiments and analysis Using the sequence of 96-FITC-labeled D-TAT derivative transporter and using the materials and methods described above, the FITC-labeled TAT-derived transporter construct into cells of the HL-60 cell line Further time-dependent internalization (uptake) was performed. These sequences are shown below.

  In the table above, D amino acids are indicated by a lowercase “d” preceding each amino acid residue (eg, dR = D-Arg).

  For some sequences, unfortunately, synthesis by the first approach failed for technical reasons. However, the remaining sequences were used for internalization experiments.

  All transporters with the consensus sequence rXXXrXXXr (see above for selection of possible sequences) showed higher internalization ability than the L-TAT transporter. Hela cells were incubated for 24 hours with 10 mmol / L (mM) r3-L-TAT-derived transporter in 96-well plates. Alternatively, human lymphoma cells are used. The cells were then washed twice with acidic buffer (0.2 mol / L (M) glycine, 0.15 mol / L (M) NaCl, pH 3.0) and twice with PBS. Cells were disrupted by adding RIPA lysis buffer. After reading the fluorescence intensity of each extract (Fusion Alpha plate reader; PerkinElmer), the background was reduced to determine the relative amount of internalized peptide.

  One position appears to be important for improving the best transporter activity and transporter activity kinetics, which is Y at position 2 (peptide N91 corresponding to SEQ ID NO: 111). Briefly, 2 in a 24-well plate with increasing doses of r3-L-TAT-derivative transporter (0.500 nmol / L (nM), 1 mmol / L (mM), or 10 mmol / L (mM)). Hela cells were incubated for hours, 6 hours, or 24 hours. The cells were then washed twice with acidic buffer (0.2 mol / L (M) glycine, 0.15 mol / L (M) NaCl, pH 3.0) and twice with PBS. Cells were disrupted by adding RIPA lysis buffer. After reading the fluorescence intensity of each extract (Fusion Alpha plate reader; PerkinElmer), the background was reduced to determine the relative amount of internalized peptide.

The conclusions of this experiment are as follows:
• After 24 hours of incubation, all transporters with the consensus sequence rXXXrXXXr (see Tables 1-3 for possible sequence selections) showed higher internalization ability than the L-TAT transporter. These results fully demonstrate the effectiveness of the consensus sequence rXXXrXXXr.
• One position affects transporter activity, which is Y at position 2 (sequence 91 corresponding to SEQ ID NO: 111).
One position has an effect on improving transporter activity kinetics, which is Y at position 2 (sequence 91 corresponding to SEQ ID NO: 111).

  Therefore, in particular, when the sequence according to the general formula (I) is 9aa, the TAT-derived sequences shown in Tables 1 to 3 in which position 2 is Y are preferable.

9. Measurement of intracellular concentrations of specific transporter constructs after uptake (internalization) of these peptides into U937 cells According to further experiments, specific transporter constructs after uptake (internalization) of these peptides into U937 cells Concentration was measured. Experiments with the sequences RKKRRQRRR (L-TAT), rrrqrrkkr (D-TAT), rKKRQRRr (r3-L-TAT), and r Y KRrQRRr (XG-91), each at a concentration of 10 μmol / L (μM) Went.

Surprisingly, a great deal of r Y KRrQRRr (XG-91, sequence 91 corresponding to SEQ ID NO: 111) accumulates in the cell, which is predicted for the transporter construct concentration or D-TAT construct in the medium. Significantly higher than the average concentration of about 20 μmol / L (μM). This is important for transporter constructs according to general formula (I), in particular transporter constructs comprising the TAT-derived sequences shown in Tables 1 to 3, in which position 2 is Y and preferably has 9aa and the consensus sequence rXXXrXXXr Emphasizes sex.

10. Peptide uptake into cells (internalization) and peptides in cell lines HepG2 (hepatocellular carcinoma), HCT-116 (neoplastic colon), U937 (lymphoma), WBC cell line (leukocyte line), and non-WBC cell lines Measurement of internalization In these experiments, in additional cell lines HepG2 (hepatocellular carcinoma), HCT-116 (neoplastic colon), U937 (lymphoma), WBC cell line (white blood cell line), and non-WBC cell line in vitro. The internalization (uptake) ability of the FITC-labeled TAT-derived transporter construct was evaluated using a fluorescent plate reader.

Test Samples and Conditions Used in Experiments The constructs and conditions used in this experiment are as described above for Experiment 3, but the following changes and cell lines were used.

a) Uptake (internalization) of FITC-labeled TAT-derived transporter construct in vitro (10 μmol / L (μM), HepG2 hepatocellular carcinoma, HCT-116 neoplastic colon, 24 hours)
The constructs used each have a length of 9 amino acids, but differ in D- / L-pattern, D-TAT and r3-TATi (also called r3-L-TATi), different TAT-derived transporter constructs called D-TAT And the constructs r ₆ R ₃ (rrrRRRRrrr) and DAK, which are further labeled at the N-terminus with β-alanine. Construct D-TAT and _r of 6 _{R 3} uptake is most efficient, then _r 3 -L-Tati was efficient.

b) Incorporation of FITC-labeled TAT-derived transporter construct in vitro (10 μmol / L (μM), U937, lymphoma, 24 hours) (internalization)
The constructs used each have a length of 9 amino acids, but differ in the D- / L-pattern, and four different TAT-derived transporter constructs (L-TAT, r3-TAT (also called r3-L-Tat), r3- TATi (also referred to as r3-L-TATi) and D-TAT). In addition, the peptide-free construct DAK was used as a control sample for comparison. Uptake of r3-TAT, r3-L-TATi, and D-TAT transporter constructs into cells was the most efficient, and uptake of L-TAT into cells was significantly less.

c) HSPG dependence of D-TAT transporter construct uptake (internalization) Experiments were conducted to determine whether uptake of D-TAT transporter construct (internalization) was HSPG-dependent. As found, D-TAT transporter construct uptake (internalization) is HSPG-dependent in U937 cells (lymphoma) at a concentration of 500 nm over 24 hours. The construct used in the experiment was D-TAT (SEQ ID NO: 4), which was 9 amino acids long, labeled with FITC, and labeled with β-alanine at the N-terminus.

d) Withdrawal of FITC-labeled TAT-derived transporter construct in U937 cells (lymphoma) Further experiments were performed to determine whether the FITC-labeled TAT-derived transporter construct was detached from U937 cells. As a result, 500 nmol / L (nM) FITC-D-TAT showed no withdrawal in U937 cells. The construct used in the experiment was 9 amino acids long, was labeled with FITC, and was D-TAT labeled with β-alanine at the N-terminus.

  Furthermore, withdrawal of the FITC-labeled TAT-derived transporter construct was observed with 10 μmol / L (μM) FITC-D-TAT, and it was found that U937 cells (lymphoma) are HSPG-dependent. The construct used for the experiment was also the D-TAT in this experiment.

e) Uptake (internalization) and escape of FITC-labeled TAT-derived transporter construct of 10 μmol / L (μM) FITC-D-TAT in non-WBC strain (white blood cell line) In further experiments, FITC-labeled TAT-derived transporter Construct uptake (internalization) and escape was seen with 10 μmol / L (μM) FITC-D-TAT in non-WBC strains (leukocyte cell lines). The construct used in the experiment was D-TAT (SEQ ID NO: 4), which was 9 amino acids long, labeled with FITC, and labeled with β-alanine at the N-terminus.

f) Conclusion As a result of the above uptake (internalization) experiment, the uptake (internalization) of a FITC-labeled TAT-derived transporter construct containing D-amino acids or consisting only of D-amino acids has been in vitro for several hours. And was found to be linear. Furthermore, after 24 hours, the uptake (internalization) of these FITC-labeled TAT-derived transporter constructs in vitro reaches an intracellular concentration 50 to 100 times higher than that of L-TAT. Furthermore, the uptake (internalization) of FITC-labeled TAT-derived transporter constructs containing D-amino acids or consisting only of D-amino acids by the WBC strain (leukocyte cell strain) is 10 times that of non-WBC strains in vitro. It was found to be ˜50 times more efficient. All these experiments showed that withdrawal was efficiently performed at high intracellular concentrations in WBC, but no withdrawal was seen at low concentrations.

11. Synthesis of cytotoxic transporter-loading conjugate molecules D-Tat-cisplatin, r3-L-TAT-cisplatin, and r3-L-TATi-cisplatin 11.1 Peptide synthesis D-TAT (rrrrqrrkkr) with further methionine, The peptide sequences of r3-L-TAT (rKKRrQRRr) and r3-L-TATi (rRRQrRKKr) are manually synthesized on 0.4 mmol Fmoc-amide-AM resin by using the Fmoc method. The peptide is then cleaved from the resin using TFA, filtered under reduced pressure, precipitated with cold ether and dried. The crude peptide is purified by semi-preparative HPLC and characterized by ESI-MS.

11.2 Alkylation of peptides to cisplatin 5.0 μmol cisplatin (1.5 mg in 3.0 mL sodium chloride buffer, pH 5.0) was added to 10 mmol / L (mM) Na ₂ HPO ₄ buffer (pH 7. 4) Dissolve in 2.0 mL and the pH value of the solution is 7.0. 5.0 μmol of D-TAT-methionine peptide (or r3-L-TAT-methionine peptide, or r3-L-TATi-methionine peptide) in 10 mmol / L (mM) Na ₂ HPO ₄ buffer (pH 7.4) The pH value of the solution is 6.0. The alkylation is then initiated by mixing the two solutions at room temperature under dark conditions (the pH value of the mixture is 7.0). After 0 h, 1 h, 3 h and 24 h, the product is analyzed by analytical PR-HPLC and characterized by ESI-MS. The predicted peak solution is finally purified by semi-preparative HPLC and lyophilized.

12 Synthesis of cytotoxic transporter-loading conjugate molecules D-TAT-oxaliplatin, r3-L-TAT-oxaliplatin, and r3-L-TATi-oxaliplatin

12.1 Synthesis of peptides (D-Tat-methionine, r3-L-TAT-methionine, and r3-L-TATi-methionine) By using the Fmoc method, on the 0.23 mmol Fmoc-Rink amide resin, D -Manually synthesize peptide sequences of TAT (rrrrqrrkkr), r3-L-TAT (rKKRrQRRr), and r3-L-TATi (rRRQrRKKr). Thus, using a cycle of Fmoc-deprotection (20% piperidine in DMF) and amino acid coupling (activation of HBTU / HOBt / DIEA in DMF), the C-terminal Gly to the N-terminal l-Met (L-type) Each amino acid up to) is continuously bound to the resin. The peptide is cleaved from the resin using TFA (2 hours in the presence of 2.5% dH ₂ O, 0.5% EDT, and 2.0% TIS), filtered at atmospheric pressure and N ₂ Reduce volume by bubbling, precipitate with cold ether and air dry. The crude peptide is purified by semi-preparative RP-HPLC and characterized by ESI-MS.

12.2 Alkylation of Peptides to Oxaliplatin In 5.0 mL of 10 mmol / L (mM) Na ₂ HPO ₄ buffer (pH 7.4), 10 μmol of oxaliplatin was added to Eloxatin® (Oxaliplatin 4. 0 mg, lactose monohydrate 36.0 mg). in dH ₂ O5.0mL, prepared D-TAT-Methionine peptide 10.0Myumol (or r3-L-TAT-Methionine peptide, or r3-L-TATi- methionine peptide) a. The alkylation is initiated by mixing the two solutions at room temperature. The reaction was then left at 37 ° C. and monitored by analytical PR-HPLC at 214 nm and 280 nm for 24 hours, target peaks were characterized by ESI-MS, purified by semi-preparative HPLC, and then lyophilized. Let

12.3 Test conditions Increasing concentrations of the conjugate molecule of the invention (D-Tat-oxaliplatin, r3) for survival of MDF-7 (human breast adenocarcinoma cell line) and SiHa (human cervical squamous cell carcinoma cell line) -L-TAT-oxaliplatin or r3-L-TATi-oxaliplatin) to determine the effect of treatment. The effects of D-Tat-oxaliplatin, r3-L-TAT-oxaliplatin, or r3-L-TATi-oxaliplatin, conjugated L-Tat-oxaliplatin, and two unconjugated anticancer drugs (oxaliplatin and cisplatin) ). Cells from each cell line (10,000 cells per well) were transferred to a 96-well plate (total volume 200 μL, for MCF-7, 10% FBS, 1% L-glutamine, 1% sodium pyruvate, MEM supplemented with% non-essential amino acids; for SiHa cells, 10% FBS, 1% sodium pyruvate, MEM / Eagle supplemented with 1% non-essential amino acids). 6 to 10 different concentrations are tested for each test substance. Control cells are not treated. Cells are incubated for 24 hours at 37 ° C. and then treated with test substances. Each experiment is performed in triplicate. Cell incubation after treatment is performed at 37 ° C. for 96 hours. The effect of the test molecule on the survival of these cell lines (in vitro cytotoxic activity) is measured by MTT assay. Add 20 μL of 5 mg / mL thiazolyl blue tetrazolium bromide (MTT, Sigma, reference number 88415) -phosphate buffered saline (PBS, CHUV) filtered through a 0.22 μm filter to each well, The plate is incubated for 4 hours at 37 ° C. The supernatant is removed and the formazan crystals are dissolved in DMSO (200 μL per well). Absorbance (OD) is measured with a microplate reader (Expert Plus Reader, Asys Hitech) at 595 nm. The IC _{50 of the} test substance (drug concentration that inhibits 50% of cell proliferation) is calculated using Prism software.

13. Synthesis of cytotoxic transporter-loading conjugate molecules D-Tat-chlorambucil, r3-L-TAT-chlorambucil, and r3-L-TATi-chlorambucil

13.1 Synthesis of conjugate molecules (D-Tat-chlorambucil, r3-L-TAT-chlorambucil, and r3-L-TATi-chlorambucil) D-TAT (rrrrqrrkkr), r3-L-TAT (rKKRrQRRr), or r3 -L-TATi (rRRQrRKKr) peptide sequence is manually synthesized on 0.23 mmol Fmoc-Rink amide resin by using Fmoc method. Thus, using a cycle of Fmoc-deprotection (20% piperidine in DMF) and amino acid coupling (activation of HBTU / HOBt / DIEA in DMF), a C-terminal Gly to an N-terminal l-A (L-type) Each amino acid up to) is continuously bound to the resin.

Following Fmoc-deprotection of N-terminal l-A (20% piperidine in DMF), chlorambucil coupling is performed using standard amino acid coupling conditions (activation of HBTU / HOBt / DIEA in DMF). The conjugate molecule is cleaved from the resin using TFA (70 minutes in the presence of 3% dH ₂ O and 3% TIS), filtered at atmospheric pressure, reduced in volume by N ₂ bubbling, and cooled with cold ether. Allow to settle and air dry. The crude conjugate molecule is purified by semi-preparative RP-HPLC, characterized by ESI-MS and then lyophilized.

13.2 Comparative Experiments Increasing concentrations of D-Tat-chlorambucil, r3-L-TAT-chlorambucil for survival of MDF-7 (human breast adenocarcinoma cell line) and SiHa (human cervical squamous cell carcinoma cell line), or The effect of treatment with r3-L-TATi-chlorambucil is determined. The effect of D-Tat-chlorambucil, r3-L-TAT-chlorambucil, or r3-L-TATi-chlorambucil was further increased with conjugated L-Tat-chlorambucil, and two u-chlorambucil unconjugated anticancer drugs (chlorambucil and cisplatin). Compare. Cells from each cell line (10,000 cells per well) were transferred to a 96-well plate (total volume 200 μL, for MCF-7, 10% FBS, 1% L-glutamine, 1% sodium pyruvate, MEM supplemented with% non-essential amino acids; for SiHa cells, 10% FBS, 1% sodium pyruvate, MEM / Eagle supplemented with 1% non-essential amino acids). 6 to 10 different concentrations are tested for each test substance. Control cells are not treated. Cells are incubated for 24 hours at 37 ° C. and then treated with test substances. Each experiment is performed in triplicate. Cell incubation after treatment is performed at 37 ° C. for 96 hours. The effect of the test molecule on the survival of these cell lines (in vitro cytotoxic activity) is measured by MTT assay. Add 20 μL of 5 mg / mL thiazolyl blue tetrazolium bromide (MTT, Sigma, reference number 88415) -phosphate buffered saline (PBS, CHUV) filtered through a 0.22 μm filter to each well, The plate is incubated for 4 hours at 37 ° C. The supernatant is removed and the formazan crystals are dissolved in DMSO (200 μL per well). Absorbance (OD) is measured with a microplate reader (Expert Plus Reader, Asys Hitech) at 595 nm. The IC _{50 of the} test substance (drug concentration that inhibits 50% of cell proliferation) is calculated using Prism software.

14 Synthesis of cytotoxic transporter-loading conjugate molecules D-Tat-doxorubicin, r3-L-TAT-doxorubicin, and r3-L-TATi-doxorubicin

14.1 Synthesis of Conjugate Molecules (D-Tat-Doxorubicin, r3-L-TAT-Doxorubicin, and r3-L-TATi-Doxorubicin) D-TAT (rrrrqrrkkr), r3-L-TAT (rKKRrQRRr), and r3 -L-TATi (rRRQrRKKr) peptide sequence is manually synthesized on 0.23 mmol Fmoc-Rink amide resin by using Fmoc method. Thus, using a cycle of Fmoc-deprotection (20% piperidine in DMF) and amino acid coupling (activation of HBTU / HOBt / DIEA in DMF), the C-terminal Gly to the N-terminal l-E (L-type) Each amino acid up to) is continuously bound to the resin.

  N-terminal l-E Fmoc-deprotection (20% piperidine in DMF) followed by acetylation (acetic anhydride, activation of DIEA in DMF). The Odmab side chain protecting group is removed using 2% hydrazine monohydrate in DMF. Coupling of chlorambucil formulated as Adriblastin® (Doxorubicin, HCl 18%, NaCl 82%, lyophilized) is performed using OBt ester (activation of DIPCDI / HOBt / DIEA in DCM / DMF).

The conjugate molecule is cleaved from the resin using TFA (2 hours in the presence of 1.7% dH ₂ O and 1.7% TIS), filtered at atmospheric pressure and reduced in volume by N ₂ bubbling. Precipitate with cold ether and air dry. The crude conjugate molecule is purified by semi-preparative RP-HPLC, characterized by ESI-MS and then lyophilized.

15. Synthesis of cytotoxic transporter-loading conjugate molecules D-Tat-Saquinavir, r3-L-TAT-Saquinavir, and r3-L-TATi-Saquinavir

15.1 Synthesis of peptides (D-Tat-D-cysteine, r3-L-TAT-D-cysteine, and r3-L-TATi-D-cysteine) By using the Fmoc method, 0.40 mmol of Fmoc-Rink On the amide resin, peptide sequences of D-TAT (rrrrqrrkkr), r3-L-TAT (rKKRrQRRr), and r3-L-TATi (rRRQrRKKr) are manually synthesized. Thus, from the C-terminal D-Arg to the N-terminal D-Cys using a cycle of Fmoc-deprotection (20% piperidine in DMF) and amino acid coupling (activation of TBTU / HOBt / DIEA in DMF) Each amino acid is continuously bound to the resin. Cleave the peptide from the resin with TFA and preincubate on ice (5 hours in the presence of 2.5% dH ₂ O, 2.5% EDT, and 1.0% TIS) and under reduced pressure , Filtered with cold ether and dried in vacuo. The crude peptide is purified by semi-preparative RP-HPLC and characterized by ESI-MS.

15.2 Preparation of the active ester of saquinavir 375 μmol of Boc-Gly-OH was dissolved in anhydrous DCM at room temperature, to which 265 μmol of DMAP, 375 μmol of DIPCI, and 110 μmol of Invirase® (lactose, excipens pro compress) Saquinavir formulated as obducto) was added at 0 ° C. The reaction mixture is warmed to room temperature and stirred overnight. The product was washed with 0.1 N HCl, dried over MgSO ₄ and evaporated under reduced pressure to give the solid product SQV-Gly (Boc). The Boc protecting group is removed by incubating the SQV-Gly (Boc) ester in a mixture of CH ₂ Cl ₂ and TFA (50:50) for 3 hours. The product is recrystallized from cold ether and dried overnight under vacuum. 47 μmol of SQV-Gly ester is dissolved in 3 mL of anhydrous DMSO at room temperature, and 94 μmol of SPDP is added thereto. The pH of the reaction mixture is adjusted to 8.0 with constant stirring at room temperature. The reaction is left for 3 hours with constant stirring. The crude product SQV-Gly-COCH2CH2-SS-pyridyl is purified by semi-preparative HPLC and characterized by ESI-MS.

15.3 Conjugates of peptide D-TAT (rrrrqrrkkr), r3-L-TAT (rKKRrQRRr), or r3-L-TATi (rRRRQrRKKR) -D-cysteine to saquinavir 27 μmol SQV-Gly-COCH2CH2-SS-pyridyl Is dissolved in 0.5 mL of PBS buffer (pH 7.5) at room temperature, and 54 μmol of D-TAT (rrrrqrrkkr), r3-L-TAT (rKKRrQRRr) in 0.5 mL of PBS buffer (pH 7.5) is dissolved therein. Alternatively, r3-L-TATi (rRRQrRKKr) -D-cysteine is added. The reaction is left at room temperature for 3 hours with constant stirring. Crude conjugates D-TAT (rrrrqrrkkr) -Saquinavir, r3-L-TAT (rKKRrQRRr) -Saquinavir, and r3-L-TATi (rRRQrRKKr) -Saquinavir are purified by semi-preparative HPLC and characterized by ESI-MS. .

16. Intracellular transfer of a transporter-loading conjugate molecule of the present invention comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200 The ability of a transporter-loading conjugate molecule comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200 to enter cells. To evaluate. The transporter constructs of the invention and the transporter-loading conjugate molecules of the invention are labeled by adding a glycine residue conjugated to fluorescein at the N-terminus. These labeled peptides (1 μmol / L (μM)) are added to the TC-3 cell culture. At a predetermined time, the cells are washed with PBS, fixed in ice-cold methanol-acetone (1: 1) for 5 minutes, and then observed with a fluorescence microscope. Fluorescein labeled BSA (1 μmol / L (μM), 12 mol / mol BSA) is used as a control.

  Fluorescent signals from these transporter constructs and the transporter-load of the present invention

17. Radiation-induced by the transporter-load conjugate molecule of the present invention comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200 Inhibition of pancreatic beta cell death JNK is also activated by ionizing radiation. A transporter-load conjugate molecule of the present invention comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200 is radiation-induced In order to determine whether to provide protection against sexual JNK injury, D-TAT, L-TAT, and a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116, and any of SEQ ID NOs: 117-200 In the presence or absence of a transporter-loading conjugate molecule of the present invention comprising a JNK1 or IB1-derived cargo peptide according to (added 1 μmol / L (μM) 30 minutes before irradiation) (30 Gy). Control cells (CTRL) are not irradiated. Cells are analyzed 48 hours later using PI and Hoechst 3342 staining as described above. N = 3. The SEM is specified.

18. Against ionizing radiation by a transporter-load conjugate molecule of the present invention comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200 Radiation protection Radiation of a transporter-loading conjugate molecule of the present invention comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200. To determine the protective effect, C57B1 / 6 mice (2 months to 3 months old) were irradiated with a Phillips RT 250 R-ray at a dose of 0.74 Gy / min (17 mA, 0.5 mm Cu filter). Irradiate. 30 minutes prior to irradiation, the transporter-load of the present invention comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200 Conjugated molecules to animals i. p. Inject. Briefly, the mouse is irradiated as follows: With the head out of the box, place the mouse in a small plastic box. The animal is placed on its back under the irradiator and the neck is secured to a small plastic tunnel to maintain the head in the correct position. Protect your body with lead. Before irradiation, mice are maintained on a standard pelleted mouse diet, but after irradiation, the semi-fluid diet is changed daily and given to the mice. Then, according to the scoring system developed by Parkins et al. (Parkins et al, Radiotherapy & Oncology, 1: 165-173, 1983), the labial mucosa reaction is scored by two independent observers. In addition to the erythema state, it evaluates the presence of edema, desquamation, and exudation. In addition, each animal is weighed before recording erythema / edema status.

19. Treatment of noise trauma TAT-derived transporter according to any of SEQ ID NOs: 1-116 contained in 2 μL of 2.6% buffered hyaluronic acid (Hylumed, Genzyme Corp.) gel formulation at a concentration of 100 μmol / L (μM) Transporter-loading conjugate molecule of the invention comprising a construct and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200, in particular the peptide inhibitor L-TAT-IB1 (s1-34) or D-TAT -A transporter-loading conjugate molecule of the invention comprising IB1 (s1-34) was added to 3 groups of guinea pigs (30 min before, 30 min, or 4 h after noisy trauma (120 kHz 6 min 30 min)). It was applied to the round window membrane of the cochlea of 6 animals in each group. Untreated ears serve as controls. Changes in auditory threshold are assessed by measuring auditory brainstem response 20 minutes after noisy trauma (transient threshold change, TTS) and 15 days after trauma (permanent threshold change, PTS). Administration of D-TAT-IB1 (s) protected against permanent hearing loss even when applied after exposure to excessive noise compared to untreated ears.

20. Transporter-load conjugate of the present invention comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200 in the treatment of colitis Evaluation of therapeutic activity of gate molecules a) Test system:
i) Species / strain: mouse / BALB / c
ii) Supplier: Harlan Israel, Ltd.
iii) Sex: female iv) Total number of animals: n = 150
v) Age: young adult, 7 weeks old at the start of the experiment vi) Body weight: the difference in body weight of the animals at the start of the treatment does not exceed ± 20% of the average body weight vii) Animal health: the health of the animals used in this experiment Status is assessed on arrival and only animals in good health are acclimatized to laboratory conditions (at least 7 days) and used for experiments.
viii) Randomization: Animals were randomly assigned to experimental groups according to a random number table.
ix) Termination: Animals alive at the end of the experiment are euthanized by cervical dislocation.

b) Test procedure Colitis was induced by administering TNBS dissolved in 50% ethanol.
All animals were then treated with a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 in doses ranging from 0.1 μg / kg to 1,000 μg / kg as single or repeated daily doses (above). And a transporter-loading conjugate molecule of the invention comprising a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200, and intraperitoneally or subcutaneously.

c) Observations and examinations i) Clinical signs During the experimental period, clinical examinations were carefully performed and recorded. For example, the appearance of skin, hair, eyes, mucous membranes, the occurrence of secretion and excretion (eg, diarrhea), and changes in autonomous activity were observed. In addition to changes in response to walking, posture, and handling, abnormal behavior, tremors, convulsions, sleep, and occurrence of coma were also recorded.

ii) Body weight Individual body weights of animals were measured daily.

iii) Clinical evaluation of colitis Body weight, stool stiffness, and transrectal bleeding were all recorded daily and used as parameters for disease severity scores.

iv) Macroscopic pathology of the colon On the last day of the experiment, the animals were euthanized and the colon was removed for macroscopic pathological evaluation according to the following scores.

21. The present invention comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200 in the treatment of chronic obstructive pulmonary disease (COPD). Of transporter-loading conjugate molecules of TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and SEQ ID NO: 117-200 in the treatment of chronic obstructive pulmonary disease (COPD) In order to determine the activity of the transporter-loading conjugate molecules of the invention comprising JNK1 or IB1-derived cargo peptides, these inventive transporter-loading conjugate molecules can be used for bleomycin-induced acute lung inflammation and fibrosis. Used in animal models. Protocols for inducing inflammation and fibrosis with bleomycin have already been described in the literature. The purpose of the experiment is
For bronchoalveolar lavage (BAL) and pulmonary neutrophil mobilization in bleomycin-induced inflammation and fibrosis on day 1 after a single dose of bleomycin (10 mg / kg) and on day 10 when fibrosis developed The purpose was to investigate the effects of these transporter-loading conjugate molecules of the present invention by the subcutaneous (sc) route.

1) Method and experimental approach Two single doses of TAT-derived transporter construct according to SEQ ID NO: 1-116 and JNK1 or IB1 according to SEQ ID NO: 117-200 together with a single intranasal bleomycin administration A transporter-loading conjugate molecule of the invention comprising a derived cargo peptide and a vehicle control are designated s. c. The mice were analyzed after 1 day and 10 days after dosing. Ten C57BL / 6 mice (8 weeks old) per group were used in this model. The experimental group comprises a vehicle, 0.001 mg / kg TAT-derived transporter construct according to any of SEQ ID NOs: 1-116, and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200. Transporter-loading conjugate molecules and 0.1 mg / kg of these transporter-loading conjugate molecules of the invention, and the treatment comprises repeated subcutaneous administration of these inventive transporter-loading conjugate molecules. Followed by administration of bleomycin once every 3 days. At 24 hours, acute lung inflammation was monitored by BAL lavage, cytodiagnosis, cell count, and lung myeloperoxidase activity. The effect of the compound was compared to the vehicle control. Ten days after a single dose of bleomycin, pulmonary fibrosis was assessed histologically using hematoxylin and eosin staining.

1.1) Bleomycin Administration A bleomycin sulfate-saline solution (10 mg / kg body weight) or physiological saline manufactured by Bell Laboratories (Montrouge, France) by instilling a volume of 40 μL under light ketamine-ketamine anesthesia into the nasal cavity. Water was administered. Bleomycin administration for both bleomycin-induced inflammation and fibrosis included: vehicle, 0.001 mg / kg TAT-derived transporter construct according to any of SEQ ID NOs: 1-116, and SEQ ID NOs: 117-200. A transporter-loading conjugate molecule of the invention comprising a JNK1 or IB1-derived cargo peptide according to any of the above and 0.1 mg / kg of the transporter-loading conjugate molecule of the invention. The route of administration for bleomycin-induced inflammation was the subcutaneous (sc) route, administered as a single dose. The route of administration for bleomycin-induced fibrosis was the subcutaneous (sc) route, administered three times over 10 days.

1.2) Bronchoalveolar lavage fluid (BALF)
After incision of the trachea, a plastic cannula was inserted and the air cavity was washed with 0.3 mL of PBS solution and heated to 37 ° C. The collected sample was divided into two fractions: the first fraction (1 mL corresponding to the first two washes) was used for mediator measurement and the second fraction was used for cell determination. (4 mL). The first fraction was centrifuged (600 g for 10 minutes), the supernatant was fractionated and stored at −80 ° C. until mediator measurement. The cell pellet was then resuspended in 0.4 mL sterile NaCl (0.9%), stored with the second fraction, and used for cell counting.

1.3) Lung homogenate After BAL, all lungs were removed and placed in a microtube (Lysing matrix D, Q Bio Gene, Illkrich, France) with 1 mL of PBS and Fastprep® system (FP120, Q Bio). Gene, Illkrich, France), and then the whole lung tissue extract is then centrifuged, and the extract is centrifuged at −80 ° C. until mediator measurement and collagen assay using Sircol Collagen Assay (France Biochem Division, France) The supernatant was saved.

1.4) Cell number and determination The total cell number in the BAL fluid was determined using a Malassez hemacytometer. After staining with MGG Diff-quick (Dade Behring AG), a differential cell count was performed on cytospin specimens (Cytospin3, Thermo Shandon). Differential cell counts were performed on 200 cells using standard morphological criteria.

1.5) TNF measurement The TNF concentration in BALF was measured using an ELISA assay kit (Mouse DuoSet, R & D system, Minneapolis, USA) according to the manufacturer's instructions. Results are reported in pg / mL.

1.6) MPO measurement The transporter-load conjugate of the present invention comprising the TAT-derived transporter construct according to any one of SEQ ID NOs: 1-116 and the JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200. MPO concentration was measured at the time of gate molecule administration.

1.7) Histology After BAL and pulmonary perfusion, large leaves were fixed with 4% buffered formaldehyde for standard microscopic analysis. 3-m sections were stained with hematoxylin and eosin (H & E).

22. Transporter-load conjugate of the present invention comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 in the treatment of Alzheimer's disease and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200 Measurement of Molecular Activity Exemplary transport of the present invention comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 in Alzheimer's disease and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200 To measure the activity of body-loading conjugate molecules, in addition to the Morris water maze behavioral test, an immunohistological test to measure plaque mass, and β-amyloid _1-40 and β- in mouse brain using an ELISA test that measures the amyloid _1-42 weight London type and Sweden In APP751 overexpressing hAPP transgenic mouse models with mutations was evaluated these peptides.

a) Method i) Introduction A TAT-derived transporter construct according to any one of SEQ ID NOs: 1 to 116 and any one of SEQ ID NOs: 117 to 200 using a female hAPP Tg mouse of 5 months (± 2 weeks) This experiment was designed to evaluate the efficacy of the transporter-loading conjugate molecules of the invention comprising such JNK1 or IB1-derived cargo peptides against behavioral, biochemical and histological markers. Therefore, mice were treated once every 2 or 3 weeks for up to 4 months and behavior was assessed in the Morris water maze at the end of the treatment period. At the time of sacrifice, brain, CSF, and blood were collected. The amounts of Aβ40 and Aβ42 in the CSF of four different brain homogenate fractions and Tg mice were measured. The amount of plaque in the cortex and hippocampus of 8 Tg mice per treatment group was quantified.

ii) Animals May (± 2 weeks) old female Tg mice with a background of C57BL / 6xDBA were randomly assigned to treatment groups 1-3 (n = 12). Starting at 5 months of age, for up to 4 months, the vehicle or two different concentrations of the TAT-derived transporter construct according to SEQ ID NO: 1-116 and the JNK1 or IB1 according to any of SEQ ID NO: 117-200 The transporter-load conjugate molecules of the invention containing the derived cargo peptide were administered subcutaneously (sc) to animals once every two or three weeks. All animals used in this experiment had dark eyes and were considered to perceive landmarks outside the MWM pool. However, for all animals, including those left in the cage for the experiment, low vision animals that were examined in visual platform training prior to the start of treatment, so-called preliminary tests, had to be excluded. If visual impairment was confirmed in a particular animal, the mouse was excluded from the experiment.

iii) Identification and rearing of animals Mice were individually identified by ear marks. Mice were housed in individual ventilation cages (IVC) on standard rodent bedding supplied by Rettenmeier®. A maximum of 5 mice were housed in each cage. Mice were maintained according to JSW standard operating procedures (SOP GEN011) written according to international standards. Each cage was identified by a colored card indicating the experiment number, gender, animal identification number (IRN), date of birth, and date of screening, and treatment group assignment. During the experiment, the temperature was maintained at about 24 ° C. and the relative humidity was maintained at about 40% to 70%. Animals were housed under a constant light cycle (12 hours light / dark). The animals were able to drink regular tap water freely.

iv) Treatment Forty (40) female hAPP transgenic mice for 4 months, two different doses of TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and any of SEQ ID NOs: 117-200 Using the transporter-loading conjugate molecule of the present invention comprising such a JNK1 or IB1-derived cargo peptide, 0.1 mg / kg b. w. Or 10 mg / kg once every 3 weeks b. w. Treatment (n = 12 / group) or vehicle once every 3 weeks. c. Processed (n = 12).

v) Morris Water Maze (MWM)
Morris water maze (MWM) test was performed in a black circular pool with a diameter of 100 cm. The tap water was filled with a temperature of 22 ± 1 ° C., and the pool was virtually divided into four zones. A transparent platform (8 cm diameter) was placed about 0.5 cm below the water surface. During the entire test period, excluding preliminary tests, the platform was located in the southwest quadrant of the pool. The day before the 4-day training period, the animals had to perform a so-called “preliminary test” (2 trials for 60 seconds) to ensure that each animal had normal vision. Only animals that performed this task were used in the MWM test. In the MWM task, each mouse had to perform 3 trials for 4 consecutive days. One trial lasted for a maximum of 1 minute. During this time, the mice had the opportunity to find a hidden transparent target. If the animal was unable to find a “way” from the water, the investigator guided the mouse to the platform or placed it on the platform. After each trial, the mouse was allowed to rest on the platform for 10-15 seconds. During this time, the mice had an opportunity to look around and confirm their position. In order not to adversely affect the tracking system (Kaminski; PCS, Biomedical Research Systems), the investigation was performed under dim light conditions. A poster with black bold geometric symbols (eg, circles and squares) is affixed to the wall around the pool, and the mouse should use that symbol as a landmark to locate it. I was able to. One swimming group per trial consisted of 5 to 6 mice to ensure a trial interval of about 5 minutes to about 10 minutes. Escape latency (time required to find the platform where the mouse is hidden and escape from the water (seconds)), path (length of the trajectory to reach the target (meters)) and in the target quadrant A computerized tracking system was used to quantify the residence time. A computer was connected to a camera located in the upper center of the pool. The camera detected the signal of a light emitting diode (LED) fixed to a small hairpin with a mouse tail. One hour after the last trial on day 4, the mice had to perform a so-called probe trial. At this time, the platform was removed from the pool and during a one minute probe trial, the experimenter counted the number of times the target had previously passed through. Furthermore, the stay time in this quadrant and the stay time in the other three quadrants were calculated. Through this trial, the mouse was unable to get any clues from the platform no matter what properties it had.

vi) Tissue sampling All surviving mice (n = 28) were sacrificed at the end of the treatment period and after all behavioral tests. Therefore, all mice were sedated by standard inhalation anesthesia (Isofluran, Baxter) as described in SOP MET030. Cerebrospinal fluid (CSF) was obtained by blunt dissection and exposure of the large occipital foramen. Upon exposure, a Pasteur pipette was inserted at a depth of about 0.3 mm to 1 mm in the large occipital foramen. CSF was collected by aspiration and capillary action until the flow stopped completely. Two aliquots of each sample were immediately frozen and stored at −80 ° C. until ready for further analysis using ELISA techniques. After sampling the CSF, each mouse was placed in the dorsal position, the thorax was opened, and a 26 gauge needle attached to a 1 cc syringe was inserted into the right ventricle. The needle was gently aspirated and blood was collected in EDTA and used to obtain plasma. To obtain plasma, rotate each mouse blood sample at 1,750 rpm (700 g) for 10 minutes in a centrifuge (GS-6R Beckman) using a rotor with swing bucket (GH-3.8 Beckman). I let you. Plasma was frozen and stored at −20 ° C. until further analysis. After sampling the blood, the hearts of the transgenic mice were perfused with 0.9% sodium chloride. The brain was quickly removed and the cerebellum was excised. The right hemisphere of all mice was fixed by immersing in freshly made 4% paraformaldehyde / PBS (pH 7.4) for 1 hour at room temperature. Thereafter, the brain was transferred to a 15% sucrose PBS solution and immersed for 24 hours to ensure cryoprotection. The next day, brains were frozen in isopentane and stored at −80 ° C. until used for histological investigation (SOP MET042). The left hemisphere was weighed, frozen in liquid nitrogen and stored at −80 ° C. for biochemical analysis.

Measurements four different brain homogenate fractions of each Tg mouse vii) A [beta] _1-40 and A [beta] _1-42, and A [beta] _1-40 and A [beta] _1-42 levels in CSF samples were assessed using an ELISA technique . High sensitivity Aβ _1-40 and Aβ _1-42 ELISA test kits were purchased from The Genetics Company ™, Switzerland (SOP MET058). CSF was prepared as described above. Frozen hemispheres of brain homogenate were homogenized in TRIS buffered saline (TBS) -buffer (5 mL) containing a protease inhibitor cocktail. 1.25 mL of this initial brain TBS homogenate was stored at −80 ° C., and 1.25 mL was further examined. The remaining brain homogenate (2.5 mL) was centrifuged, and the resulting supernatant (= TBS fraction) was dispensed and stored at −20 ° C. until ELISA measurement. The pellet was suspended in Triton X-100 (2.5 mL), centrifuged, and the supernatant (= Triton X-100 fraction) was dispensed and stored at −20 ° C. These steps were repeated using SDS (2.5 mL). The pellet of the SDS fraction was suspended in 70% formic acid (0.5 mL) and then centrifuged. The obtained supernatant was neutralized with 1 mol / L (M) TRIS (9.5 mL), dispensed, and stored at −20 ° C. (= FA fraction). Samples of 4 brain homogenate fractions (TBS, Triton X-100, SDS, and FA) were used for Aβ _1-40 and Aβ _1-42 measurements using ELISA technology. The ELISA test kit was purchased from The Genetics Company ™, Switzerland (SOP MET062). It could be inferred that TBS and Triton X-100 solubilized the monomer-oligomer structure. Polymer-like prefibrils and water-insoluble fibers could be dissolved in SDS and FA. In this regard, examining all four fractions gives insight into the polymerization state of A.

vii) Evaluation of brain morphology All Tg animal brain tissues investigated were manipulated in exactly the same way to avoid bias due to variability in this procedure. Twenty frozen sections (Leica CM 3050S) per layer (5 layers in total) each having a thickness of 10 μm were cut in a sagittal manner from half of the brains of 24 Tg mice (8 mice in each group). Sheets (one from each layer) were processed and evaluated to quantify the amount of plaque. The five sagittal layers are shown in FIGS. 104-105, 107-108, 111-112, 115-116, and 118-118 of the morphological atlas "The Mouse Brain" (2nd edition) by Paxinos and Franklin. Corresponded to 119. The first layer was identified by the requirement to include the entire hippocampus with regions CA1, CA2, CA3, GD1b, and GDmb. Immunoreactivity was quantitatively assessed in the hippocampus and cortex using monoclonal human Aβ-specific antibody 6E10 (Signet) and thioflavin S staining. The remaining brain hemisphere or tissue that was not used was stored in JSW CNS until the end of the project.

b) Evaluation i) Behavior In the Morris water maze trial, the length of the swimming distance, the escape latency, the swimming speed, the number of times the platform passed through the previous position in the probe trial, and the staying time in each quadrant of the pool Measured for each Tg animal using special computer software.

ii) Biochemical evaluation All Tg mouse CSF samples and brain specimen samples were analyzed using commercially available Aβ _1-40 and Aβ _1-42 ELISA. Appropriate standard measurements were performed simultaneously. Brain specimen samples were analyzed in duplicate. Since there was only a small amount, the CSF sample could only be measured once.

iii) Histology il) Measurement of amyloid deposition and plaque mass The following evaluation procedure was used for 6E10 immunohistochemistry:
aa) Contrast images to visualize segment boundaries without applying contrast to the images.
bb) Interactive drawing of the cortical outline and measuring the cortical area (= area of the area).
cc) Interactive drawing of the area of interest (AOI), where a stained object is detected that exceeds a certain intensity and is greater than 8 μm ² based on a threshold level (same for each image) .
dd) Measure the number of objects after smooth contrast to increase the area of each object, the sum of the stained areas in the AOI, and the signal / noise ratio (same for each image).
ee) Repeat aa) to dd) for the hippocampus.
ff) Average plaque size (= “total plaque area / number of plaques”), relative plaque number and area (= “number of plaques / region area” and “total plaque area / region area × 100”) calculate.
gg) Automatically export data including parameters “image title, area area, number of plaques, total area of plaques, relative number of plaques, relative plaque area, and average plaque size” to an Excel spreadsheet. The image quality and exclusion criteria were recorded, respectively, using the findings field. Exclusion criteria were missing sections, wrinkled, large scratches, or staining inconsistencies (eg, due to swelling that could hinder adequate reaction of the blocking reagent).
hh) Close the image without saving (to save the raw data as is).

23. The transporter of the present invention comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 in the treatment of type 2 diabetes and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200 Measurement of activity of cargo conjugate molecule The purpose is to use a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 in the treatment of type 2 diabetes, a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200, and In the measurement of the activity of a transporter-loading conjugate molecule of the present invention comprising, specifically, fasting blood glucose level once every 3 days (28 days) The effect of chronic treatment with these inventive transport volume conjugation molecules in the db / db mouse model There was to be.

a) Materials and Methods i) Animals A total of 20 male db / db mice (8 weeks old) were purchased from Charles River (Germany). Upon arrival, animals are housed (n = 6-7 / group), unless otherwise specified, with regular rodent diet (Altromin standard # 1324 chow; C. Petersen, Ringsted, Denmark) and water ad libitum It was.

  Mice were housed in a temperature and humidity controlled room under a 12:12 L / D cycle (lights up at 4:00 and turns off at 16:00).

ii) Groups and randomization On day 4, mice were randomized according to blood glucose concentration (fasting; blood glucose measured using Biosen Sline analyzer (EKF diagnostic, Germany)) and the following drugs Assigned to one of the treatment groups (n = 6):
1) Vehicle control, S.I. C. (Physiological saline)
2) A transporter-loading conjugate molecule of the present invention comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200; / Kg; s. c.
3) A transporter-loading conjugate molecule of the present invention comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and a JNK1- or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200; 10 mg / Kg; s. c. .
All the above doses were calculated for the free base. The purity of the drug was 95.28% and the peptide content was 78.0%. All compounds were administered subcutaneously (sc) in a volume of 3 mL / kg. A transporter-load conjugate molecule of the present invention comprising a vehicle control and a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200. The formulation procedure was as follows.

First, a transporter-loading conjugate molecule of the present invention comprising a TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and a JNK1- or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200 is used as a vehicle. Dissolved in. The formulations (concentrations of 0.33 mg / mL and 3.3 mg / mL, corresponding to doses of 1 mg / kg and 10 mg / kg, respectively) were prepared according to the procedure detailed below. Concentrations were calculated and expressed taking into account the purity and peptide content of the test items (multiplication count was 1.346).
-Stock solution preparation: a transporter of the present invention comprising a freeze-dried TAT-derived transporter construct according to any of SEQ ID NOs: 1-116 and a JNK1 or IB1-derived cargo peptide according to any of SEQ ID NOs: 117-200 -The cargo conjugate molecule was thawed for a minimum of 1 hour and prepared as a vehicle stock solution of 1 mmol / L (mM) (corresponding to 3.823 mg / mL). Aliquots were prepared for each treatment day and stored at about -80 ° C. On each treatment day, the stock solution is diluted to the required concentration.
-Stock solution storage: about -80 ° C;
-Storage of diluted preparation: 24 hours at maximum room temperature.

  Prior to solubilization, the powder was stored at -20 ° C. The stock solution is stable at about −80 ° C. for 3 months, and the diluted formulation for administration to animals is stable for 24 hours at room temperature. Diluted material that was not used could be stored for up to 7 days when stored at 4-8 ° C.

c) Experimental procedure After acclimatization for 8 days, mice were treated once a day at AM 8:00 for 21 days by SC administration 8 hours before extinguishing at PM 4:00 according to the outline group.

i) Blood glucose Blood glucose was administered to animals fasted for 7 hours by collecting 10 μL of blood sample from the tail vein into a hematocrit tube and then analyzing with Biosen s-line analyzer (EKF-diagnostic; Germany). Blood glucose was measured 6 hours later.

ii) Metabolic cages Group 1 + 3: In addition to 24-hour food and water intake, mice were placed in metabolic cages to record 24-hour urine and fecal excretion. Mice were stratified into two subteams of n = 6-7 and then metabolic characterization was performed.

iii) Adipokine Panels Group 1 + 3: Blood was collected from the tail vein 3 times using a hematocrit tube (100 μL) coated with EDTA. After centrifuging the blood, plasma was collected and stored at −20 ° C. until measurement. The following panel of adipokines / cytokines was then measured using the Luminex line 7-plex: leptin, resistin, MCP-1, PAI-1, TNFα, insulin, and interleukin-6 (IL-6).

iv) Termination Group 1 + 3 (day 111): The following organs were excised and weighed: subcutaneous fat in the groin, epididymal fat, retroperitoneal fat, brain, liver, kidney, spleen and heart. All organ samples were fixed with 4% PFA for future histopathological evaluation. In addition, the pancreas (generally) was sampled to allow steric analysis and immunohistochemical analysis, and the eye was sampled for later analysis of retinopathy. Group 2 (Day 28): No tissue or plasma was collected.

24. TAT derivatives target human leukocyte population Primary human leukocytes (WBC) were obtained from whole blood after erythrocyte lysis. Incubate WBC with 1 μmol / L (μM) D-TAT (SEQ ID NO: 4) -FITC or r3-L-TAT (SEQ ID NO: 15) -FITC for 30 minutes at 37 ° C., wash with acidic buffer, cell type Stain with fluorescent antibodies against specific surface markers (CD14 for monocytes, CD15 for multinucleated leukocytes, CD3 for lymphocytes T, CD19 for lymphocytes B). Cells containing D-TAT-FITC and r3-L-TAT-FITC are finally analyzed by flow cytometry to determine the respective transporter content. Both TAT derivatives target the human leukocyte population. dTAT and r3LTAT bind to monocytes, neutrophils, and lymphocyte T cells, and the binding efficiency to lymphocyte B cells is low. There is a slight difference between the specificity of dTAT and r3-L-TAT, and D-TAT binds to lymphocyte T more efficiently than r3-L-TAT.

25. Incorporation of selected transporter constructs according to the invention by different cell types 96 wells pre-coated with poly-D-lysine cells of subconfluent density (which may vary depending on the cell type used) Plated on plate. Different FITC-coupled transporters were then incubated with the cells at 3 μmol / L (μM) for 15 hours. After this time, the cells were stored on ice for the rest of the procedure. In order to remove the peptides bound to the cell surface, the cells were first washed twice with an acid wash to remove molecules bound to the plasma membrane. The cells were then washed twice with PBS and lysed in standard lysis buffer for 30 minutes. The plate containing the cell lysate was then centrifuged at 1,500 rpm for 5 minutes at 4 ° C. The clear supernatant was then collected and transferred to a black 96 well plate for measurement of intracellular FITC fluorescence. The following cells were used:
White blood cell line: Raw: Macrophage cell (mouse)
J77: Macrophage cells (mouse)
Primary purified leukocytes: BMDM: Bone marrow derived macrophages (mouse).

  The results are expressed as percent incorporation of D-TAT (SEQ ID NO: 4) (FIG. 17) or r3-L-TAT (SEQ ID NO: 15) (FIG. 18). All transporter constructs show uptake into their respective cells, albeit at different rates.

26. Limb immunohistochemistry of CFA-induced inflammation (4 hours)
In this experiment, 8-week-old male C57 / B16 mice were used. Peripheral inflammation was induced by subcutaneous injection of Freund's Complete Adjuvant (CFA) (Sigma, 20 μL) into the left hind limb that was briefly anesthetized with isofluorane. Animals treated with XG-102 (SEQ ID NO: 233) received a single bolus of 10 μg / kg XG-102 one hour prior to CFA injection. v. Injected. Mice were sacrificed 4 hours after CFA injection by perfusing the heart with 4% PFA-PBS solution. The hind limbs were cryoprotected with sucrose (30% in PBS) and then frozen with liquid isopentane and stored for further frozen section preparation. Sections were stained with hematoxylin and eosin (H & E) or processed for XG-102 (SEQ ID NO: 233) immunostaining. First, the 0.3% _{H 2 O} 2 - was quenched 30 minutes sections with methanol, rinsed for three minutes with _{H 2} O, then washed for 5 minutes with PBS. After this step, sections were preincubated for 45 minutes in PBS containing 15% serum and 0.3% Triton, then diluted with 1.5% serum, 0.1% Triton in PBS. Incubated overnight with primary antibody (rabbit polyclonal anti-XG-102, 1/1000 dilution) at room temperature. Sections were then incubated with the appropriate biotinylated secondary antibody for 2 hours at room temperature, washed 3 times with PBS, and incubated with streptavidin-biotin-peroxidase complex (ABC kit, Vectastein, Vector Laboratories) for 2 hours at room temperature. . The immunolabeling was developed after washing 3 times with PBS using 2,3′diaminobenzidene as substrate diluted 1/10 in buffer according to the manufacturer (Roche). Finally, the sections were dehydrated and mounted using Eukitt (Kindler GmBH). All animals were processed for immunostaining in the same experiment and subjected to color development for the same time. HRP staining (lower panel) revealed the presence of XG-102 in infiltrating leukocytes in animals treated with CFA-XG-102.

27. CD11b staining In this experiment, 8-week-old male C57 / B16 mice were used. Peripheral inflammation was induced by subcutaneous injection of Freund's Complete Adjuvant (CFA) (Sigma, 20 μL) into the left hind limb that was briefly anesthetized with isofluorane. Animals treated with XG-102 (SEQ ID NO: 233) received a single bolus of 10 μg / kg XG-102 one hour prior to CFA injection. v. Injected. Mice were sacrificed 4 hours after CFA injection by perfusing the heart with 4% PFA-PBS solution. The hind limbs were cryoprotected with sucrose (30% in PBS) and then frozen with liquid isopentane and stored for further frozen section preparation. Sections were stained with XG-102 antibody or CD11b used as a leukocyte surface marker. First, Sections 0.3% _H 2 _{O 2} - was quenched 30 minutes in a methanol solution, rinsed for three minutes with _{H 2} O, then washed for 5 minutes with PBS. After this step, sections were preincubated for 45 minutes in PBS containing 15% serum and 0.3% Triton, then diluted with 1.5% serum, 0.1% Triton in PBS. Incubated overnight with primary antibody (rabbit polyclonal anti-XG-102, 1/1000 dilution) at room temperature. The sections are then incubated with the appropriate biotinylated secondary antibody for 2 hours at room temperature, washed 3 times with PBS, and incubated for 2 hours at room temperature with streptavidin-biotin-peroxidase complex (ABC kit, Vectastein, Vector Laboratories). did. The immunolabeling was developed after washing 3 times with PBS using 2,3′diaminobenzidene as substrate diluted 1/10 in buffer according to the manufacturer (Roche). Finally, the sections were dehydrated and mounted using Eukitt (Kindler GmBH). All animals were processed for immunostaining in the same experiment and subjected to color development for the same time.

28. Detection and kinetics of XG-102 (SEQ ID NO: 233) in rat whole blood cells Male Sprague Dawley rats weighing 180-200 g were treated with 0.1 mg / kg XG-102 (SEQ ID NO: 233) in a single bolus i.d. v. Processed by injection. The injection time is considered the reference time for the following sampling. Rats were sacrificed after various injection times of 30 minutes, 24 hours, 3 days, 7 days, 14 days, and 27 days. Rats were sacrificed by phlebotomy. The mesenteric lymph node chains (and neck, axilla, upper arm, kidney, and lumbar lymph nodes, if possible) were then removed for histological sections. Organs were rinsed with 1 × PBS at 4 ° C. and fixed overnight in 4% PAF / 1 × PBS at 4 ° C. After 24 hours, the sample was rinsed for 3 × 10 minutes in 1 × PBS at 4 ° C. with stirring and soaked in 30% sucrose / 1 × PBS until the LN subsided. The samples were then frozen with isopentane for 3 minutes at − (35-40) ° C. using vials on dry ice. After preparing frozen sections, sections for XG-102 immunostaining were processed. First, Sections 0.3% _H 2 _{O 2} - was quenched 30 minutes in a methanol solution, rinsed for three minutes with _{H 2} O, then washed for 5 minutes with PBS. The sections were then preincubated for 45 minutes in PBS containing 15% serum and 0.3% Triton, then primary antibody diluted with 1.5% serum in PBS, 0.1% Triton ( Rabbit polyclonal anti-XG-102, 1/1000 dilution) and incubated overnight at room temperature. After washing 3 times with PBS, sections were incubated with streptavidin-biotin-peroxidase complex (ABC kit, Vectastein, Vector Laboratories) for 2 hours at room temperature. The immunolabeling was developed after washing 3 times with PBS using 2,3′diaminobenzidene as substrate diluted 1/10 in buffer according to the manufacturer (Roche). The sections were then dehydrated and encapsulated using Eukitt (Kindler GmBH). All animals were processed for immunostaining in the same experiment and subjected to color development for the same time.

29. FITC-XG-102 (SEQ ID NO: 233) and FITC-D-TAT (SEQ ID NO: 4) localization to liver and lymph nodes 1 mg / kg FITC-XG102 (SEQ ID NO: 233) was added to the tail of female Sprague Dawley rats. ) Or 1 mg / kg FITC-D-TAT (SEQ ID NO: 4) i. v. Injected. Six hours after injection, rats were sacrificed with a lethal dose of sodium pentobarbital (150 mg / kg ip). For histological analysis, the rat heart was perfused with 4% paraformaldehyde (PFA) -PBS solution during anesthesia. Organs (liver and lymph nodes) were cryoprotected with sucrose (30% in PBS), frozen with liquid isopentane and stored for further frozen section preparation. 14 μm sections were incubated in PBS for 5 minutes and then Hoechst stained (1 μg / mL) for 2 minutes at room temperature. After washing with PBS, the sections were enclosed in Fluorsave inclusion medium. Sections were visualized using an epifluorescence microscope.

Claims (15)

A transporter-loading conjugate molecule for transporting a cargo molecule to a white blood cell, wherein the transporter-loading conjugate molecule comprises:
a) As a component (A), a (poly) peptide consisting of the amino acid sequence represented by any of SEQ ID NO: 15 and SEQ ID NO: 16 ;
b) As component (B), a cargo molecule;
c) one or more optional ingredients;
Transporters, characterized in including things - cargo conjugate molecule. Use of a ( poly) peptide for producing a transporter-loading conjugate molecule for transporting a cargo molecule to a leukocyte, wherein the (poly) peptide is represented by any one of SEQ ID NO: 15 and SEQ ID NO: 16. The use characterized by consisting of the amino acid sequence .   2. The transporter-load conjugate molecule of claim 1, wherein two or more components of the transporter-load conjugate molecule are covalently bonded to each other.   4. The transporter-load conjugate molecule of claim 3, wherein components (A) and (B) are covalently bonded to each other. The cargo molecule (component (B)) is
a) a protein or (poly) peptide comprising at least one of a therapeutically active protein and a therapeutically active (poly) peptide,
b) a protein kinase inhibitor comprising an inhibitor of c-Jun amino terminal kinase or a factor that is a protein kinase;
c) an antigen,
d) antibodies,
e) an apoptotic factor,
f) a protease inhibitor involved in a medical condition comprising a peptide protease inhibitor;
g) BH3-domain,
h) BH3-only protein,
i) DNA,
j) RNA, including siRNA, antisense RNA, microRNA,
k) cytotoxic agents,
l) small organic compounds,
m) small molecule drugs,
n) gold particles,
o) fluorescent dyes,
p) antibiotics,
The transporter-loading conjugate molecule according to any one of claims 1 , 3, and 4 , wherein q) is selected from at least one of a static virus agent.   From any of claims 1 and 3, wherein any one or more further components are selected from signal sequences or localization sequences that direct transporter-loading conjugate molecules to a specific intracellular target location or a specific cell type. 6. The transporter-loading conjugate molecule according to any one of 5. From the amino acid sequence encoded by the nucleotide sequence represented by any one of SEQ ID NO: 117 and SEQ ID NO: 120, SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 121-200, The transporter-loading conjugate molecule according to any one of claims 1 and 3 to 6, which is an inhibitor of c-Jun amino terminal kinase consisting of a selected amino acid sequence. 8. Transporter-loading conjugate molecule according to any of claims 1 and 3 to 7, wherein transport to leukocytes occurs ex vivo. The transporter-loading conjugate molecule according to any one of claims 1 and 3 to 8, wherein the leukocytes are primary cells. The transporter-loading conjugate molecule according to any one of claims 1 and 3 to 9, wherein the leukocytes are immortalized cells. The transporter-loading conjugate molecule according to any one of claims 1 and 3 to 10, wherein the leukocytes are transgenic cells. The transporter according to any one of claims 1 and 3 to 11, wherein the leukocytes are selected from at least one of granulocytes, lymphocytes, monocytes, macrophages, dendritic cells, microglia cells, and mast cells. Cargo conjugate molecule. 13. A transporter-loading conjugate molecule according to claim 12, wherein the granulocytes are selected from the group consisting of neutrophils, eosinophils, and basophils. 13. A transporter-loading conjugate molecule according to claim 12, wherein the lymphocytes are selected from NK cells, helper T cells, cytotoxic T cells, [gamma] [delta] T cells, and B cells. An isolated leukocyte comprising a transporter-load conjugate molecule or fragment thereof, wherein the transporter-load conjugate molecule comprises:
a) As a component (A), a (poly) peptide consisting of the amino acid sequence represented by any of SEQ ID NO: 15 and SEQ ID NO: 16;
b) As component (B), a cargo molecule;
c) one or more optional ingredients;
An isolated leukocyte characterized by comprising: